QuantStudio™ Design and Analysis Desktop

Software
USER GUIDE

Getting started with design and analysis of experiments in the

desktop software v1.5.x

for use with:

QuantStudio™ 1 Real-Time PCR System
QuantStudio™ 3 Real-Time PCR System
QuantStudio™ 5 Real-Time PCR System
Publication Number MAN0010408
Revision C.0

For Research Use Only. Not for use in diagnostic procedures.

 Life Technologies Holdings Pte Ltd | Block 33 | Marsiling Industrial Estate Road 3 | #07-06, Singapore 739256
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL,
INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT,
INCLUDING YOUR USE OF IT.
Revision Date Description
™
C.0 19 October 2018 Updated to add QuantStudio 1 Real-Time PCR System information.
B.0 December 2015 Updates include:
• Combination of define and assign functions into a single Plate tab
™
• Display of VeriFlex Zones on plate layout
• Real-time data monitoring in the Run tab
• Security, Audit, and E-Signature (SAE) features
• Implementation of locked workflow
• Selection of an instrument before starting a run
• Ability to select multiple targets in the results view
• Various minor changes to the user interface

A.0 April 2015 New document.

NOTICE TO PURCHASER: DISCLAIMER OF LICENSE: Purchase of this software product alone does not imply any license under any process,
instrument or other apparatus, system, composition, reagent or kit rights under patent claims owned or otherwise controlled by Life Technologies
Corporation, either expressly, or by estoppel.
Trademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered
trademark of Roche Molecular Systems, Inc., used under permission and license.
©2018 Thermo Fisher Scientific Inc. All rights reserved.
 Contents

■ CHAPTER 1 Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
™
Features in the QuantStudio Design and Analysis Desktop Software . . . . . . . . . . . . . . . . . 10
Experiment types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Workflow overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

■ CHAPTER 2 General procedures to set up and run experiments . . . 14

Set global desktop software preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Workflow: Set up and run an experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Set up a template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Create or open a template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Enter template properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Confirm or edit the run method and optical filter selection . . . . . . . . . . . . . . . . . . . . . . 17
Assign plate and well attributes using the Quick Setup subtab . . . . . . . . . . . . . . . . . . . 18
Save a template file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Prepare reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Good laboratory practices for PCR and RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Guidelines for handling samples and reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Guidelines for setting up the reactions in the plates or tubes . . . . . . . . . . . . . . . . . . . . 21
Start and monitor a run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
View real‑time run information in the desktop software . . . . . . . . . . . . . . . . . . . . . . . . . 23
View real‑time run information on the instrument touchscreen . . . . . . . . . . . . . . . . . . 23
View the Post‑Run Summary in the desktop software . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Experiment setup using libraries, external files, and templates . . . . . . . . . . . . . . . . . . . . . . 24

■ CHAPTER 3 General procedures to review results . . . . . . . . . . . . . . . . . . 26

About the quantification cycle (Cq) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Overview of the Results tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Workflow: General procedures to review the run results . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Guidelines for viewing and analyzing results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Assess results in the Amplification Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Amplification Plot overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Assess the overall shape of the Amplification Plot curves . . . . . . . . . . . . . . . . . . . . . . . 30
Confirm or correct threshold settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

QuantStudio™ Design and Analysis Desktop Software User Guide 3

Contents

Confirm or correct baseline settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Omit outliers from analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Optimize display of negative controls in the Amplification Plot . . . . . . . . . . . . . . . . . . . 34
Assess results in the Well Table view . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Well Table overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Group or sort the Well Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Review the dye signal profile using the Multicomponent Plot . . . . . . . . . . . . . . . . . . . . . . . . 36
Multicomponent Plot overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
View and assess the Multicomponent Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Review the signal profile using the Raw Data Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Raw Data Plot overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
View and assess the Raw Data Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Review the flags in the QC Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
View calibration results in the desktop software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Review ROI/Uniformity calibration results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Review Background calibration results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Review Dye calibration results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Override the calibration data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Export experiments or results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Options for exporting run data and results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Export configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

■ CHAPTER 4 Set up, run, and review standard curve experiments . 41

Standard curve experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Reaction types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Compatible PCR options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Set up a standard curve experiment in the software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Set up and run the PCR reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Review results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Workflow: Review standard curve experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Standard Curve Plot overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
View and assess the Standard Curve Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Standard curve settings overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

4 QuantStudio™ Design and Analysis Desktop Software User Guide

 Contents

■ CHAPTER 5 Set up, run, and review

relative standard curve experiments and
comparative Ct experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Relative standard curve experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Reaction types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Compatible PCR options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Comparative Ct experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Reaction types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Compatible PCR options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Relative quantitation: relative standard curve vs. comparative Ct . . . . . . . . . . . . . . . . . . . . . 51
Set up a relative standard curve experiment in the software . . . . . . . . . . . . . . . . . . . . . . . . . 51
Set up a comparative Ct experiment in the software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Set up and run the PCR reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Review results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Workflow: Review relative standard curve and comparative Ct experiments . . . . . . . 54
View and assess the Standard Curve Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Gene Expression Plot overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
QC Plot overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Relative quantification settings overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

■ CHAPTER 6 Set up, run, and review genotyping experiments . . . . . . 60

Genotyping experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Reaction types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Compatible PCR options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Set up a genotyping experiment in the software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Set up and run the PCR reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Review results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Workflow: Review genotyping experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Allelic Discrimination Plot overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
View and assess the Allelic Discrimination Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Perform manual calls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Call settings overview (genotyping) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

QuantStudio™ Design and Analysis Desktop Software User Guide 5

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■ CHAPTER 7 Set up, run, and review

presence/absence experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Presence/absence experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Reaction types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Compatible PCR options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Set up the presence/absence experiment in the software . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Set up and run the PCR reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Review results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Workflow: Review presence/absence experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Presence/Absence Plot overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
View and assess the Presence/Absence Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Call settings overview (presence / absence) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

■ CHAPTER 8 Set up, run, and review melt curve experiments . . . . . . 75

Melt curve experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Reaction types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Set up a melt curve experiment in the software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Set up melt curve reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Review results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Workflow: Review melt curve experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Melt Curve Plot overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
View and assess the Melt Curve Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Melt curve settings overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

■ APPENDIX A Instrument overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

Power on the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
View run history and delete or transfer files from the instrument . . . . . . . . . . . . . . . . . . . . 80
Load and unload the plate in the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
™
Load and unload a plate in the QuantStudio 1 Real-Time PCR Instrument . . . . . . . . 81
™
Load and unload a plate in the QuantStudio 3 Real-Time PCR Instrument or
™
QuantStudio 5 Real-Time PCR Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

6 QuantStudio™ Design and Analysis Desktop Software User Guide

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■ APPENDIX B Alternative procedures to set up a template . . . . . . . . . . 83

Set up a custom experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Custom experiments overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Set up a custom experiment in the software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
Assign samples using a sample definition file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
About sample definition files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Create a sample definition file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Import sample information from a sample definition file . . . . . . . . . . . . . . . . . . . . . . . . 85
Assign samples and targets using plate setup files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
About plate setup files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Import plate setup data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Assign targets, samples, and biological replicate groups from an XLS file . . . . . . . . . . . . . 87
Create new EDT files using existing EDT and EDS files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
About experiment templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

■ APPENDIX C Detailed procedures to create or edit a method . . . . . . 88

Adjust method parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
™
Set up advanced temperature zones (Auto Delta and VeriFlex Zones) . . . . . . . . . . . . . . . . 89
Add or adjust a pause step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Select optical filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Edit the ramp increment for the melt curve dissociation step . . . . . . . . . . . . . . . . . . . . . . . . 91

■ APPENDIX D Detailed procedures to set up

plate / well details and libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Assign well attributes (Quick Setup subtab) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Define and assign well attributes (Advanced Setup subtab) . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Assign a task to wells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Detection tasks for targets and SNP assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Define and set up standard dilutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Assign the standard dilutions manually . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Define and assign biological replicate groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Sample, target, and SNP assay libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
Libraries overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
Apply a filter to search a library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
Define samples in the Samples table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Define targets in the Targets table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Define SNP assays in the SNP Assays table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

QuantStudio™ Design and Analysis Desktop Software User Guide 7

Contents

■ APPENDIX E Configure analysis settings . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

Guidelines for the analysis settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
View and configure the analysis settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Ct settings overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Flag settings overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Advanced settings overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

■ APPENDIX F Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . 103

™
Related documentation for the QuantStudio 1 Real-Time PCR System . . . . . . . . . . . . . . 103
™
Related documentation for the QuantStudio 3 Real-Time PCR System and
™
QuantStudio 5 Real-Time PCR System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

8 QuantStudio™ Design and Analysis Desktop Software User Guide

 1 Product information

■ Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
■ Features in the QuantStudio™ Design and Analysis Desktop Software . . . . . . . 10
■ Experiment types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
■ Workflow overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Product description
The QuantStudio™ Design and Analysis Desktop Software allows the user to open,
run, and analyze experiments generated with QuantStudio™ 1 Real-Time PCR System,
QuantStudio™ 3 Real-Time PCR System, and QuantStudio™ 5 Real-Time PCR System.
The software also allows you to set up experiments, send experiments to the
instrument, collect data, and analyze the collected data.

QuantStudio™ Design and Analysis Desktop Software User Guide 9

 1 Chapter 1 Product information
Features in the QuantStudio™ Design and Analysis Desktop Software

Features in the QuantStudio™ Design and Analysis Desktop Software

Actions Unlocked template Locked template[1,2]

Properties tab

Edit experiment name; enter / scan plate barcode; enter user name 3 3

Select instrument / block type, experiment type, chemistry, run mode 3 —

Enter reagent information (chemistry details) 3 3

Method tab

Edit the thermal protocol, reaction volume, optical filter selection 3 —

Plate tab

Quick Setup subtab

Define plate attributes 3 —

Define or assign samples 3 3

Define or assign targets or SNP assays 3 —

Advanced Setup subtab

Define samples 3 3

Assign samples 3 3

Define targets or SNP assays 3 —

Assign targets or SNP assays 3 3

Run tab

Start and monitor a run in progress 3 3

View time remaining and plots[3] 3 3

Results tab

Review run results (analyzed run data) 3 3

Configure analysis settings 3 —

Export tab

Select export options for run data and run results (analyzed run data) 3 3

Export run data[3]; export template settings 3 3

[1] If you enter the password for a locked template, all listed actions are available.
[2] Always save a backup unlocked version of a template before saving it as a locked template.
[3] This feature is also available from the instrument touchscreen.

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 Chapter 1 Product information
Experiment types 1

Experiment types
Purpose Description

Standard curve experiment

Determines absolute 1. The software measures amplification of the target in a standard dilution series and in
target quantity in test samples.
samples. 2. The software generates a standard curve using data from the standard dilution series.
3. The software uses the standard curve to interpolate the absolute quantity of target in
the test samples.

Relative standard curve experiment

Determines relative 1. The software measures amplification of the target of interest and of an endogenous
target quantity in control target in a standard dilution series, in a reference (calibrator) sample, and in
samples. test samples.
The endogenous control is a target that is expressed equally in all samples; examples of
endogenous controls are β‐actin, GAPDH, and 18S ribosomal RNA. The software can
algorithmically incorporate multiple endogenous control targets in relative
quantification calculations.
The reference sample is used as the basis for relative quantification results (or
1× sample). For example, in a study of drug effects on gene expression, an untreated
control is an appropriate reference sample.
2. The software generates standard curves for the target of interest and the endogenous
control using data from the corresponding standard dilution series.
3. The software uses the standard curves to interpolate the quantities of the target of
interest and the endogenous control in each sample. The target quantity in each sample
is then normalized to the sample's endogenous control quantity.
4. To determine the relative quantity of the target in test samples, the software divides the
normalized target quantity in the sample by the normalized target quantity in the
reference sample.

Comparative Ct (DDCt) experiment

Determines relative 1. The software measures amplification of the target of interest and of an endogenous
target quantity in control target in a reference (calibrator) sample and in test samples.
samples. The endogenous control is a target that is expressed equally in all samples; examples of
endogenous controls are β‐actin, GAPDH, and 18S ribosomal RNA. The software can
algorithmically incorporate multiple endogenous control targets in relative
quantification calculations.
The reference sample is used as the basis for relative quantification results (or
1× sample). For example, in a study of drug effects on gene expression, an untreated
control is an appropriate reference sample.
2. The measurements for the target of interest are normalized to the endogenous control.
3. To determine the relative quantity of the target in test samples, the software compares
the normalized ΔCq (ΔCt or ΔCrt) for the sample to the normalized ΔCq (ΔCt or ΔCrt) for
the reference sample.

QuantStudio™ Design and Analysis Desktop Software User Guide 11

1 Chapter 1 Product information
Experiment types

Purpose Description

Genotyping experiment
Detects single Genotyping experiments use preformulated TaqMan® SNP Genotyping Assays that include
nucleotide the following components:
polymorphism (SNP)
• Two sequence-specific primers for amplification of sequences containing the SNP of
variants of a target
interest
nucleic acid
sequence. • Two allele-specific TaqMan® probes for Allele 1 and Allele 2
1. The software normalizes the fluorescence of the reporter dyes to the fluorescence of
the passive reference dye in each well.
2. The software plots the normalized reporter dye signal of each sample well on an Allelic
Discrimination Plot, which contrasts the reporter dye intensities of the allele-specific
probes.
3. The software algorithmically clusters the sample data, and assigns a genotype call to
the samples of each cluster according to its position on the plot.

Presence/absence experiment
Determines the The software calls the target present or absent based on an algorithmically determined call
presence or absence threshold. (The call threshold is different from the Ct threshold; the Ct threshold is not used
of a target nucleic to make calls.)
acid sequence in a
sample.

Melt curve experiment

Determines the In the software, melt curve analysis is included in the default run method for any experiment
melting temperature type that uses intercalating dyes.
(Tm) of the
amplification 1. The software plots a melt curve based on the fluorescence of the dye with respect to
products of a PCR change in temperature.
that used 2. Using the melt curve, the software calculates the melting temperature (Tm).
intercalating dyes.

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 Chapter 1 Product information
Workflow overview 1

Workflow overview
Use the tabs across the top of the screen to navigate the workflow in the
QuantStudio™ desktop software.

Create a template (EDT file)

Set up template parameters (properties, method, plate and well details)

Configure analysis settings

Run the EDT file on the instrument

Review run results (EDS file)

QuantStudio™ Design and Analysis Desktop Software User Guide 13

 2 General procedures to set up and
run experiments

■ Set global desktop software preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

■ Workflow: Set up and run an experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
■ Set up a template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
■ Prepare reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
■ Start and monitor a run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
■ Experiment setup using libraries, external files, and templates . . . . . . . . . . . . . . 24

Set global desktop software preferences

1. In the menu bar, select Tools4Preferences.

2. Set preferences in each tab.

Tab Options Action
Defaults Decimal Places to Show Enter the number of significant figures in exported results.
Language [if available] Choose a language for the software from the dropdown menu.
Experiment Baseline Start Cycle and Enter the first and last cycles to be used to calculate the baseline
Baseline End Cycle for runs that include amplification.
Auto Analysis Select to perform auto analysis at the end of each run.
Auto Save Select to save changes at the end of each run.
Print Disable color when printing the Select to disable color printing.
Well Table
Export Use Last File Location or Use Select where to export results.
Default Folder Click Browse, then navigate to and select a default location.
Display Date Format, Time Format, and Select the display formats. These formats are also used in the
Format Decimal Point Format export or import of data.

3. Click Save.

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 Chapter 2 General procedures to set up and run experiments
Workflow: Set up and run an experiment 2

Workflow: Set up and run an experiment

Set up a template (page 15)

Create or open a template (page 16)

Enter template properties (page 16)

Confirm or edit the run method and optical filter selection (page 17)

Assign plate and well attributes (page 18)

Save a template file (page 20)

Prepare reactions (page 21)

Start and monitor a run (page 22)

For post-run procedures, see the following sections:

• Chapter 3, “General procedures to review results“
• “Export experiments or results“ on page 40

Set up a template
This section describes the general procedures to set up a template in the desktop
software. For setup information for a specific experiment type, see the following
sections.
• “Set up a standard curve experiment in the software“ on page 43
• “Set up a relative standard curve experiment in the software“ on page 51
• “Set up a comparative Ct experiment in the software“ on page 53
• “Set up a genotyping experiment in the software“ on page 61
• “Set up the presence/absence experiment in the software“ on page 69
• “Set up a melt curve experiment in the software“ on page 76
• “Set up a custom experiment in the software“ on page 84
You can also use features of the desktop software to more easily set up some or all of
an experiment. For example, you can set up an experiment using one of the following
strategies.
• Use desktop software libraries to set up samples, targets or SNP assays, run
methods, and analysis settings (see “Sample, target, and SNP assay libraries“ on
page 96).
• Import experiment parameters from external files or templates (see “Experiment
setup using libraries, external files, and templates“ on page 24).

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2 Chapter 2 General procedures to set up and run experiments
Set up a template

Create or open a Create a new template or open an existing template in the Home screen.
template • In the New Experiment pane, perform one of the following tasks to create a
new template.
To Action
Create a template Click Create New Experiment.
without
preexisting
settings
Create a template 1. Select Create New Experiment4Template.
from a system 2. Navigate to and select the desired file, then click Open.
template
System template files are installed with the software in:
<drive>:\ Program Files (x86)\Applied
BioSystems\QuantStudio Design & Analysis
Software\templates ,
where <drive> is the drive on which the software is
installed.

• In the Open Existing Experiment pane, perform the following tasks to open
an existing template.
a. Click Open.

b. Navigate to and select the desired file, then click Open.

Enter template 1. Click the Properties tab to open and edit the experiment properties.
properties
2. (Optional) In the Name field, modify the file name.
The Name field determines two file names:
• The initial EDT file name.
Note: After the initial EDT file save, modifying the Name field does not
update the EDT file name. To change the EDT file name after the initial save,
select Save4Save As.
• The default file name for the EDS file created during an instrument run.

3. (Optional) Click the Barcode field, then scan or enter a plate barcode.

4. (Optional) Enter information in the User name and the Comments fields, if
applicable.

5. Select an Instrument type, Block type, Experiment type, Chemistry (reagents),

and Run Mode (Fast or Standard cycling) from the dropdown lists.
Note: The experiment type defines the available options for the template setup.
For more information on the parameters defined in each experiment type, see
“Experiment definitions“ on page 20 .

6. (Optional) Click Manage chemistry details (see “Enter reagent information“).

7. (Optional) Click Save or select Save4Save as.

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 Chapter 2 General procedures to set up and run experiments
Set up a template 2

Enter reagent information

1. In the Properties tab, click Manage chemistry details.

2. Click Add.

3. Enter the reagent type, name, part number, lot number, and expiration date.

4. (Optional) Add a custom attribute for a reagent.

a. Click in the table header to add a column for a custom attribute.

b. Click the Custom Attribute column header, then enter a new attribute.

c. Select a cell in the Custom Attribute column, then enter its information.

d. (Optional) Click in the header to delete a custom attribute from the table.

5. (Optional) Click to delete a reagent from the table.

6. Click Close.

Scan a barcode using the optional barcode scanner

The instrument is compatible with an optional Handheld Barcode Scanner
(Cat. No. 4488442, purchased separately). The barcode scanner reads
Code 128 (alphanumeric), which supports 128 ASCII character barcodes.

1. Click the Barcode field.

2. Hold the scanner 20–30 cm away from a plate or container label and aim at the
center of the barcode, then press the trigger.

3. Slowly move the scanning beam across the barcode until the scanner emits a
high-pitched tone.

When the scanner scans a barcode, it automatically transmits the following

information:
• Transmits the alphanumeric equivalent of the barcode to the barcode field.
• Transmits other reagent information (Lot #, Part #, Expiration Date, etc.)
For more information about the hand-held barcode scanner, see the user
documentation provided with the barcode scanner.

Confirm or edit In the Method tab, perform the follow tasks if needed.
the run method • (Optional) Adjust the reaction volume.
and optical filter
selection • (Optional) Edit the default run method (thermal protocol).
– The default run method is optimized for TaqMan® assays and a broad range
of other reagents.
– To edit the default run method, see “Adjust method parameters“ on
page 88.

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2 Chapter 2 General procedures to set up and run experiments
Set up a template

• (Optional) Edit the default optical filter selection (see “Select optical filters“ on
page 90).
– The default optical filter selection is for factory-calibrated (system) dyes.
– For more information about instrument supported dyes and their calibration
and optical filter selection, see QuantStudio™ 1 Real-Time PCR System
Installation, Use, and Maintenance Guide (Pub. No. MAN0017853) or
QuantStudio™ 3 and 5 Real-Time PCR Systems Installation, Use, and Maintenance
Guide (Pub. No. MAN0010407).

Method elements

1 2

6
8

1 Reaction volume 5 Step temperature

2 Heated cover temperature 6 Step hold time
3 Stage of thermal protocol 7 Number of cycles
4 Step within a stage 8 Temperature ramp rate

Assign plate and Note: This section provides general procedures to set up the plate.
well attributes · For detailed procedures to set up a plate, see Appendix D, “Detailed procedures to
using the Quick set up plate / well details and libraries“.
Setup subtab · For specific instructions for each experiment type, see the corresponding chapter in
this guide.

1. In the Plate tab, select plate wells in the Plate Layout or the Well Table.
For more information, see “Select plate wells“ on page 19.

2. Click Quick Setup.

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 Chapter 2 General procedures to set up and run experiments
Set up a template 2

3. Assign the well attributes for the selected wells.

• Into the text fields, enter the sample and target or SNP assay names.
• From the dropdown lists, select a defined sample and target or SNP assay.
For more information about defining or importing samples and targets or
SNP assays, see “Define and assign well attributes (Advanced Setup subtab)
“ on page 93.
• Click Advanced Setup, then change the default selections for the reporter and
quencher dyes and for tasks where applicable. For more information, see
“Assign a task to wells“ on page 93.

4. (Optional) Enter comments for the selected wells.

5. In the Plate Attributes pane, select a Passive Reference from the dropdown list.

Select plate wells

• Select plate wells in the Plate Layout.
To Action
Select a single well Click a well in the plate
Select multiple wells Click-drag in the plate
Select contiguous wells Shift-click wells in the plate
Select non-contiguous wells Ctrl-click wells in the plate
Select a column of wells Click a column header
Select all wells Click the top-left corner of the plate grid
Select a block of wells Click a well to define a corner, then Shift-click another
well on the opposite corner
Deselect a single well Ctrl-click the selected well

• Select plate wells in the Well Table.

To Action
Select a single well Click a row in the table
Select multiple wells Click-drag in the table
Select contiguous wells Shift-click rows in the table
Select non-contiguous wells Ctrl-click rows in the table
Deselect a single well Ctrl-click the selected row

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2 Chapter 2 General procedures to set up and run experiments
Set up a template

Experiment definitions
The parameters that you can define vary by experiment type.

Biological Reference and

Passive
Experiment Type Targets SNP Assays Samples Replicate Endogenous
Reference
Groups Controls

Standard Curve

Relative Standard
Curve

Comparative Ct

Melt Curve

Genotyping

Presence /
Absence

Custom

Save a template Save a template file as an unlocked template

file Note: You cannot save a locked template as an unlocked template.

• Save the template with the same EDT file name.

• In any tab, click Save.
• In the menu bar, select File 4Save.

• Save the template with a new EDT file name.

Note: You cannot save a locked template with a new file name.
• In any tab, select Save4Save as.
• In the menu bar, select File 4Save As.

Save a template file as a locked template

IMPORTANT! Always save a backup unlocked version of a template before saving it as

a locked template.

1. In the menu bar, select File 4Save As a Locked Template.

2. Enter and confirm a password, then click OK to continue saving the file.
Note: The password is required to open the template with unlimited editing
options. Without the password, a locked template can still be opened but with
limited editing options.
Note: Record the password because lost passwords cannot be recovered.

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 Chapter 2 General procedures to set up and run experiments
Prepare reactions 2

Prepare reactions
See instrument user guide for information about compatible reagents and required
materials for PCR reactions.
Follow the instructions provided by the manufacturer to prepare reactions.
Follow the other guidelines described in this section.

Good laboratory • Wear clean gloves and a clean lab coat.

practices for PCR – Do not wear the same gloves and lab coat that you have previously used
and RT-PCR when handling amplified products or preparing samples.
• Change gloves if you suspect that they are contaminated.
• Maintain separate areas and dedicated equipment and supplies for:
– Sample preparation and reaction setup.
– Amplification and analysis of products.
• Do not bring amplified products into the reaction setup area.
• Open and close all sample tubes carefully. Avoid splashing or spraying samples.
• Keep reactions and components capped as much as possible.
• Use a positive-displacement pipettor or aerosol-resistant barrier pipette tips.
• Clean lab benches and equipment periodically with 10% bleach solution or DNA
decontamination solution.

Guidelines for • Use calibrated pipettors and aerosol-resistant tips.

handling samples • Prepare the reaction mixes according to the recommendations that are provided
and reagents by the manufacturer of the master mixes and assay mixes.
• Include excess volume in calculations to account for the loss that occurs during
reagent transfers.
• Use TE buffer or water to dilute samples and standards.
• Use care when diluting samples and standards. Mistakes or inaccuracies in
making the dilutions affect data accuracy.
• Keep the dilutions and assay mix frozen and protected from light until use.
Excessive exposure to light can affect the fluorescent probes or dyes.
• Perform the following tasks before each use:
– Mix the master mix thoroughly by swirling the bottle.
– Resuspend the assay mix by vortexing, then centrifuge the tube briefly.
– Thaw any frozen samples by placing them on ice. When thawed, resuspend
the samples by vortexing, then centrifuge the tubes briefly.

Guidelines for • Use good laboratory practices for PCR and RT-PCR. For more information, see
setting up the “Good laboratory practices for PCR and RT-PCR“ on page 21.
reactions in the • Ensure that the arrangement of the PCR reactions matches the plate layout
plates or tubes displayed in the software.

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2 Chapter 2 General procedures to set up and run experiments
Start and monitor a run

• Confirm that the liquid in each well is at the bottom of the well and free of
bubbles. If it is not, centrifuge the plate again.

• Ensure that plates or tubes are properly sealed.

• Keep the reaction plate or tubes at 4°C and protected from light until you are
ready to load the plate into the instrument.
• Keep the bottom of the plate clean. Fluids and other contaminants on the bottom
of the plate can contaminate the sample block and cause an abnormally high
background signal.
• If necessary, use a permanent marker or pen to mark a tube and the side of a
plate. Do not use fluorescent markers.

Start and monitor a run

IMPORTANT! Before loading a reaction-filled plate into the instrument, review the
detailed procedures in “Load and unload the plate in the instrument“ on page 81.

1. Go to an instrument connected to the computer that is running the desktop

software.

2. Load the plate into the instrument.

CAUTION! (QuantStudio™ 3 Real-Time PCR System and QuantStudio™ 5 Real-

Time PCR System only) The instrument should be used by trained operators
who have been warned of the moving parts hazard.

3. Open a template (EDT file) in the desktop software.

4. (Optional) In the Export tab, select Auto Export to export run results
automatically after the run ends.

5. In the Method tab and the Plate tab, review the template parameters and setup.

6. In the Run tab, select the instrument to use from the START RUN dropdown list.

7. Accept or edit the default name for the EDS file, then click Save.
Note: EDS files contain the run data and results. The system creates the EDS
default file name for the EDS file from the Experiment Name in the Properties
tab.

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 Chapter 2 General procedures to set up and run experiments
Start and monitor a run 2

8. During the instrument run, monitor the run:

• In the Run tab of the desktop software.
• In the instrument touchscreen.

9. Unload the plate from the instrument.

CAUTION! PHYSICAL INJURY HAZARD. During instrument operation,

the plate temperature can reach 100°C. Allow it to cool to room
temperature before handling.

Note: (QuantStudio™ 3 Real-Time PCR System and QuantStudio™ 5 Real-Time PCR

System only) If the instrument does not eject the plate, contact Support.

Note: If the connection between the instrument and the desktop software is
interrupted during the run, the instrument still completes the run. However, the run
data (EDS file) must be transferred from the instrument to the desktop software using
a USB drive or a network drive.

View real‑time run You can view real-time run information in the Run tab of the desktop software.
information in the • In the Run tab, view the time remaining for the run and the run status.
desktop software
• In the Run tab, in the Amplification Plot subtab, view real-time data and plots.
• Click View to select the data that are displayed in each well.
• Select wells in the plate layout to highlight respective curves in the plot.
• Select curves in the plot to highlight respective wells in the plate layout.
Note: For melt curve experiments, you can only monitor the melt curve plot.

View real‑time run You can view real-time run information in the instrument home screen.
information on the • In the instrument home screen, view the block temperature, the time remaining
instrument for the run, and the run status.
touchscreen
• Touch or swipe left once to view real-time run method information.

• Touch or swipe left twice to view real-time data and plots.

View real-time data and plots on the instrument touchscreen

1. In the instrument home screen, during an instrument run, touch or swipe left
twice.

2. Touch Well details.

3. Touch Samples, Targets, or Tasks to select a graphical representation of each

selection.

4. Touch Close to return to the home screen.

QuantStudio™ Design and Analysis Desktop Software User Guide 23

 2 Chapter 2 General procedures to set up and run experiments
Experiment setup using libraries, external files, and templates

Adjust the display of real-time plots on the instrument touchscreen

1. In the instrument home screen, during an instrument run, touch or swipe left
twice to view real-time data and plots.

2. Touch Zoom.

3. Touch or to zoom in or out.

4. Touch the arrows to pan left, right, up, or down on the graph.

5. Touch Close to return to the default view.

View the Post‑Run You can view a summary of the run after the run ends.
Summary in the In the Run tab, click the Post-Run Summary tab to view a summary of the run,
desktop software including the following information:
• Experiment Name
• User Name
• Errors Encountered
• Instrument Serial Number and Instrument Name
• Start Time, Stop Time, and Run Duration

Experiment setup using libraries, external files, and templates

The desktop software offers the following features so that you can more easily set up
some or all of an experiment using libraries, external files, and templates.
• Use desktop software libraries to set up samples, targets or SNP assays, run
methods, and analysis settings (see “Libraries overview“ on page 96).
• Import some or all of an experiment setup from external files or templates (see
the following table).

Table 1 Experiment setup from external files or templates

Option Action Setup information

Import sample information Import a sample definition file – Sample name
(define samples). (see “Assign samples using a – (Optional) Custom sample properties
sample definition file“ on
page 85).
Import samples, targets, and Import a plate setup file (see Plate setup information:
well assignments. “Assign samples and targets – Well number
using plate setup files“ on
– Sample name
page 86).
– Sample color
– Target name
– Dyes
– (Optional) Other well information

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 Chapter 2 General procedures to set up and run experiments
Experiment setup using libraries, external files, and templates 2

Option Action Setup information

Set up the plate layout in a Copy-paste from an XLS file Plate setup information:
spreadsheet without saving to a (see “Assign targets, samples, – Well number
special format. and biological replicate groups
– Sample name
or from an XLS file“ on page 87).
– Biological Group
Use a subset of the columns in
a plate layout spreadsheet. – Target name
– Task
– Dyes
– Quantity
– Comments
– (Optional) Other well information

Use a complete template setup Create a new template from an – Plate setup information, as above
from an existing EDT or EDS existing template or run results – Reagent information
file. file (see “Create new EDT files
using existing EDT and EDS – Thermal protocol
files“ on page 87). – Analysis settings

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 3 General procedures to review
results

■ About the quantification cycle (Cq) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

■ Overview of the Results tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
■ Workflow: General procedures to review the run results . . . . . . . . . . . . . . . . . . . 29
■ Guidelines for viewing and analyzing results . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
■ Assess results in the Amplification Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
■ Assess results in the Well Table view . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
■ Review the dye signal profile using the Multicomponent Plot . . . . . . . . . . . . . . 36
■ Review the signal profile using the Raw Data Plot . . . . . . . . . . . . . . . . . . . . . . . . 37
■ Review the flags in the QC Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
■ View calibration results in the desktop software . . . . . . . . . . . . . . . . . . . . . . . . . . 38
■ Export experiments or results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

This section includes information about reviewing results and configuring analysis
settings for all experiment types. For information about a specific experiment type, see
the corresponding chapter in this guide.
For step-by-step instructions for results review procedures, see the desktop software
Help.

About the quantification cycle (Cq)

Term Name Description

Cq Quantification Cq is the general form for gene expression metrics. Cq values (both Ct and Crt) are used
cycle as the primary input values for sample quantification experiments: absolute
quantification (AQ) and relative quantification (RQ).
Ct and Crt are the algorithm-specific calculations of Cq.

Ct Threshold cycle The PCR cycle number at which the fluorescence signal meets the threshold in the
amplification plot.
Ct is the gene expression metric result when using the Baseline Threshold Algorithm.

Crt Relative The PCR cycle number for the threshold calculated from the modeled amplification
threshold cycle efficiency profile.
Crt is the gene expression metric result when using the Relative Threshold Algorithm.

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 Chapter 3 General procedures to review results
Overview of the Results tab 3

Overview of the Results tab

Review and analyze run data in the Results tab. In the Results tab, two additional
tools display at the right of the workflow bar.

• Click Analyze after omitting wells or changing the analysis settings.

• Click to access analysis settings.
Note: The analysis settings and plots that are available vary by experiment type.

1 3

Figure 1 Plot pane

1 Plot toolbar 3 Expand/contract the plot pane display
2 Plot selection list (varies by experiment) 4 Plot legend

The Plot toolbar includes the following options:

• Zoom in and out • Configure plot properties
• Print or copy plot image • Show/hide plot legend
• Save plot as image file • Configure plot settings

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3 Chapter 3 General procedures to review results
Overview of the Results tab

1 3

Figure 2 Plate Layout

1 Expand/contract the Plate Layout display (layout is expanded in this figure)
2 View: Select well properties to display
3 Plate Layout toolbar

The Plate Layout toolbar includes the following options:

• Zoom in and out • Display Plate Layout
• Fit plate to window • Display Well Table

2 3

1 4

Figure 3 Well Table

1 Expand/contract the Well Table display 3 Group by: Select a parameter by which to
(table is expanded in this figure) group well rows
2 View: Select well properties to display 4 Well Table toolbar

The Well Table toolbar includes the following options:

• Expand grouped rows • Display Plate Layout
• Collapse grouped rows • Display Well Table

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 Chapter 3 General procedures to review results
Workflow: General procedures to review the run results 3

Workflow: General procedures to review the run results

When a run is complete, the desktop software automatically analyzes the run data
using the analysis settings that are specified during template development. The
software then displays the run results in the Results tab.

View the Amplification Plot to confirm or correct

threshold and baseline settings (page 30)

Assess the experiment plot for the experiment

(for example, view the Allelic Discrimination Plot for genotyping experiments)
(see the corresponding chapter in this guide)

Review data for outliers and (optional) omit wells (page 33)

(Optional) View the Multicomponent Plot to review the dye signal profile (page 36)

(Optional) View the Raw Data Plot to review the signal profile (page 37)

(Optional) Review flags in the QC Summary (page 38)

(Optional) Configure the analysis settings (page 100)

IMPORTANT! If you omit wells or configure the analysis settings, click Analyze to
reanalyze the data.

Guidelines for viewing and analyzing results

• For information about adjusting the views in the Results tab, see the desktop
software Help.
• To reanalyze the data, select all the wells in the Plate Layout, then click
Analyze.
• To enable auto-analysis of data after a run, select
Tools4Preferences4Experiment, then select Auto Analysis.

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 3 Chapter 3 General procedures to review results
Assess results in the Amplification Plot

Assess results in the Amplification Plot

Amplification Plot The Amplification Plot displays sample amplification as a function of cycle number or
overview well. You can use the amplification plot to perform the following tasks:
• Confirm or correct baseline and threshold values.
• Identify outliers.
• Identify and examine abnormal amplification. Abnormal amplification can
exhibit one of the following characteristics:
– Increased fluorescence in negative control wells.
– Absence of detectable fluorescence at an expected cycle.
Note: If you notice abnormal amplification or a complete absence of
fluorescence, see the instrument user guide for troubleshooting information.
Three plots are available. Some plots can be viewed as a linear or log10 graph.

Table 2 Amplification Plot types

Plot type Description Use to

DRn vs Cycle DRn is the magnitude of normalized • Identify and examine

fluorescence signal, relative to the irregular amplification.
baseline fluorescence, generated by • View threshold values for
the reporter at each cycle during the the run.
PCR amplification.

Rn vs Cycle Rn is the fluorescence signal from • Identify and examine

the reporter dye normalized to the irregular amplification.
fluorescence signal from the passive • View baseline values for
reference. the run.

Ct vs Well Ct is the PCR cycle number at which • Locate outlying

the fluorescence meets the amplification (outliers).
threshold in the amplification plot.

Assess the overall You can assess the overall shape of the Amplification Plot curves in the Results tab.
shape of the If no data are displayed in the Results tab, click Analyze.
Amplification Plot
curves 1. In the Results tab, select Amplification Plot from the dropdown list.

2. Click to configure the plot, then make the following selections:

• Plot Type: ΔRn vs Cycle
• Graph Type: Log
• Plot Color: Target, Sample, or Well
The Amplification Plot is displayed for the selected wells in the Plate Layout.

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 Chapter 3 General procedures to review results
Assess results in the Amplification Plot 3

4
3

Figure 4 Typical Amplification Plot

A typical amplification curve has four distinct sections:
1 Baseline 3 Linear phase
2 Exponential (geometric) phase 4 Plateau phase

Confirm or correct 1. In the Results tab, select Amplification Plot from the dropdown list.
threshold settings
2. Click to configure the plot, then make the following selections:
• Plot Type: ΔRn vs Cycle
• Graph Type: Log
• Plot Color: Target, Sample, or Well
The Amplification Plot is displayed for the selected wells in the Plate Layout.

3. (Optional) Adjust the threshold.

• Click-drag the threshold bar into the exponential phase of the curve.
• Configure the Ct analysis settings (see “Ct settings overview“ on page 101).

Table 3 Examples of threshold settings

Set the threshold in the exponential phase of the amplification curve. A threshold set above
or below the optimum can increase the standard deviation of the replicate groups.

Threshold setting
Example
evaluation

Threshold set correctly.

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3 Chapter 3 General procedures to review results
Assess results in the Amplification Plot

Threshold setting
Example
evaluation

Threshold set too low.

Threshold set too high.

Confirm or correct 1. In the Results tab, select Amplification Plot from the dropdown list.
baseline settings
2. Click to configure the plot, then make the following selections:
• Plot Type: Rn vs Cycle
• Graph Type: Linear
• Plot Color: Well
• Select Show: Baseline Start / Baseline End
Note: The start and end cycles are used to calculate the baseline.
The Amplification Plot is displayed for the selected wells in the Plate Layout.
The start ( ) and end ( ) cycles display for each well.

3. (Optional) Adjust the start and end cycle values for the baseline (see “Ct settings
overview“ on page 101).

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 Chapter 3 General procedures to review results
Assess results in the Amplification Plot 3

Figure 5 Example of correct baseline

Set the end cycle ( ) a few cycles before the cycle number where significant fluorescence
signal is detected.

Omit outliers from Outlier wells have Cq (Ct or Crt) values that differ significantly from the average for
analysis the associated replicate wells. To ensure Cq (Ct or Crt) precision, consider omitting the
outliers from analysis.

1. In the Results tab, select Amplification Plot from the dropdown list.

2. Click , then make the following selections to configure the plot:

• Plot Type: Ct vs Well
• Graph Type: Linear
• Plot Color: Well
The Ct values are displayed for the selected wells in the Plate Layout.

3. Click to examine the Well Table for outliers.

a. Select Group by4Replicate.

b. Identify outliers in each replicate group.

Outlier wells typically have one or more QC flags.

4. Omit outliers in either the Well Table or Plate Layout view.

• In the Well Table, select Omit in outlier rows of the table.
• In the Plate Layout, right-click a well, then select Omit.

5. Click Analyze to reanalyze the run data with any outliers removed.

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 3 Chapter 3 General procedures to review results
Assess results in the Amplification Plot

Optimize display 1. In the Results tab, select Amplification Plot from the dropdown list.
of negative
2. Click to configure the plot, then make the following selections:
controls in the
Amplification Plot • Plot Type: ΔRn vs Cycle
• Graph Type: Linear
• Plot Color: Target
• Deselect Show: Threshold
• Deselect Show: Baseline Start / Baseline End

3. In either the Plate Layout or Well Table, select the negative control wells
(wells that should not have amplification for a particular target).

4. Click (configure plot properties), then select the Y Axis tab.

a. Deselect Auto-adjust range.

b. Enter Minimum value of –1.

c. Enter Maximum value of 2.

d. Click Save.

Figure 6 Example Amplification Plot of negative controls

The linear plot displays the Amplification Plot for negative controls as smooth lines. The
expanded Y axis displays low levels of amplification.

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 Chapter 3 General procedures to review results
Assess results in the Well Table view 3

Assess results in the Well Table view

Well Table The Well Table displays data for each well in the reaction plate. The data that are
overview displayed depend on the specific experiment type and can include the following
information:
• Sample name, target name, task, and dyes
• Values that are specific to particular stage of the method
For example: Ct or Crt, normalized fluorescence (Rn), or melt temperature (Tm)
• Values that are specific to a particular experiment type
For example: genotype calls, presence/absence calls, or quantities
• Omitted wells
• QC flags
• Comments

Group or sort the Some of the possible options for grouping or sorting the Well Table are described
Well Table in the following table. Available grouping categories depend on the specific
experiment type and analysis settings.
Note: You can select multiple columns when sorting, but you can only make one
selection for grouping rows.

Group category Description Notes

Replicate[1,2,3] Grouped by replicate • Examine the Ct or quantity values for each

replicate group to assess the precision of Ct
values.

Flag Grouped as flagged and unflagged • A flag indicates that the software found a potential
wells error in the flagged well.
• For more information about QC flags, see the
desktop software Help.

Ct[1,2,4] Grouped by Ct value • Ct value < 8—There may be too much template in
the reaction.
• Ct value > 35—There may be a low amount of
target in the reaction; for Ct values > 35, expect a
higher standard deviation.

RQ[2] Grouped by RQ value • RQ value < 1—There is less relative target in the
test sample as compared to the calibrator sample.
• RQ value > 1—There is more relative target in the
test sample as compared to the calibrator sample.

Call[4,5] Grouped by genotype call or —

presence/absence call

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 3 Chapter 3 General procedures to review results
Review the dye signal profile using the Multicomponent Plot

Group category Description Notes

Tm1[3] Grouped by Tm value Tm1 refers to the dominant peak.

This grouping category is only applicable for the
following experiment types:
• Melt curve experiments
• Any experiment with a melt curve data collection
step (e.g., absolute standard curve)

[1] For standard curve experiments.

[2] For relative standard curve and comparative Ct (ΔΔCt) experiments.
[3] For melt curve experiments.
[4] For genotyping experiments.
[5] For presence/absence experiments.

Review the dye signal profile using the Multicomponent Plot

Multicomponent The Multicomponent Plot displays the complete spectral contribution of each dye
Plot overview over the duration of the PCR run.
Use the Multicomponent Plot to obtain the following information.
• Confirm that the signal from the passive reference dye remains unchanged
throughout the run.
• Review reporter dye signal for spikes, dips, and/or sudden changes.
• Confirm that no amplification occurs in the negative control wells.

View and assess You can view and assess the Multicomponent Plot in the Results tab.
the If no data are displayed in the Results tab, click Analyze.
Multicomponent
Plot 1. In the Results tab, select Multicomponent Plot from the dropdown list.

2. Click to configure the plot, then make the following selections:

• Plot Color: Dye
The Multicomponent Plot is displayed for the selected wells in the Plate
Layout.

3. In the Plate Layout, select wells one at a time, then examine the
Multicomponent Plot for the following plot characteristics.

Plot characteristic Description

Passive reference dye The passive reference dye fluorescence signal should remain relatively constant
throughout the PCR process.
Reporter dye The reporter dye fluorescence signal should display a flat region corresponding to
the baseline, followed by a rapid rise in fluorescence as the amplification proceeds.

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 Chapter 3 General procedures to review results
Review the signal profile using the Raw Data Plot 3

Plot characteristic Description

Irregularities in the signal Spikes, dips, and/or sudden changes in the fluorescence signal may have an impact
on the data.
Negative control wells The negative control wells should show no significant increase in fluorescence
signal.

Figure 7 Example Multicomponent Plot (single well)

Review the signal profile using the Raw Data Plot

Raw Data Plot The Raw Data Plot displays the raw fluorescence signal (not normalized) for each
overview optical filter during each cycle of the real-time PCR.
View the Raw Data Plot to confirm a stable increase in signal (no abrupt changes or
dips) from the appropriate filter.

View and assess You can view and assess the Raw Data Plot in the Results tab.
the Raw Data Plot If no data are displayed in the Results tab, click Analyze.

1. In the Results, select Raw Data Plot from the dropdown list.
The Raw Data Plot is diaplayed for the selected wells in the Plate Layout.

2. Click to display the Show Cycle scale.

3. Click-drag the Show Cycle pointer from cycle 1 to cycle 40, and confirm that
each filter displays the characteristic signal increase.
For more information on each filter set, see the instrument user guide (see
Appendix F, “Documentation and support“).

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 3 Chapter 3 General procedures to review results
Review the flags in the QC Summary

Figure 8 Example Raw Data Plot

Review the flags in the QC Summary

The QC Summary in the Results tab displays a list of the QC flags, including the flag
frequency and location.
If no data are displayed in the Results tab, click Analyze.

1. In the Results tab, select QC Summary from the dropdown list.

2. Review the Flag Details table and the summary.

The Wells column of the Flag Details table identifies wells that triggered a flag.

3. (Optional) In the Flag Details table, click each flag to display a brief description of
the flag.
For more information about the flag, see the desktop software Help.

View calibration results in the desktop software

Calibration results obtained on the instrument can be transferred via USB to the
desktop software.

1. In the Home screen, click Open.

2. Navigate to and select the desired calibration EDS file.

If you are viewing calibration data files, Calibration QC Status is displayed to the
right of the Plate Layout and Well Table.
Note: For more information about using the Plate Layout and Well Table, see
Chapter 3, “General procedures to review results“ .

Review 1. In the ROI tab, select a Filter Set from the dropdown list to see the
ROI/Uniformity corresponding results.
calibration results 2. In the Uniformity tab, use the Plate Layout, Well Table, and
Calibration QC Status to review the run results.

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 Chapter 3 General procedures to review results
View calibration results in the desktop software 3

Review 1. Select the plate wells in the Plate Layout or the Well Table to view the
Background corresponding curves. For more information, see “Select plate wells“ on page 19.
calibration results 2. Review data in the Well Table.
a. Review the results for each well in tabular format.

b. Sort the wells according to well or normalized fluorescence with each filter.

c. Select wells to review data in the analysis plot.

3. Click Calibration QC Status to review the quality of the calibration data.

Review Dye 1. Select a Dye row in the Calibration table to view the corresponding analysis data
calibration results plot.

2. Select the plate wells in the Plate Layout or the Well Table to view the
corresponding curves. For more information, see “Select plate wells“ on page 19.

3. Review data in the Well Table.

a. Review the results for each well in tabular format.

b. Sort the wells according to well or normalized fluorescence with each filter.

c. Select wells to review data in the analysis plot.

4. Click Calibration QC Status to review the quality of the calibration data.

Override the Each EDS file contains the calibration data from the instrument on which it was run.
calibration data You can use calibration data from another instrument for analysis of your run data.
• Calibration data must be from the same block type (96-well 0.2-mL block,
96-well 0.1-mL block, or 384-well block).
• Calibration data for a QuantStudio™ 1 Real-Time PCR Instrument run must be
from a QuantStudio™ 1 Real-Time PCR Instrument run.
• Calibration data from a QuantStudio™ 5 Real-Time PCR Instrument run can
override calibration data for a QuantStudio™ 3 Real-Time PCR Instrument run.
• Calibration data from a QuantStudio™ 3 Real-Time PCR Instrument run can not
override calibration data for a QuantStudio™ 5 Real-Time PCR Instrument run.

1. Open the EDS file to recalibrate.

2. Select Analysis4Override Calibration4Use Calibration From Another File.

3. Navigate to, then select the EDS file containing the alternative calibration data.

4. Click Open.

5. (Optional) To revert to the original calibration data, select Analysis4Override

Calibration4Revert To Original Calibration.

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3 Chapter 3 General procedures to review results
Export experiments or results

Export experiments or results

For step-by-step instructions for exporting experiments or results, see the desktop
software Help.

Options for To Action

exporting run data
and results Save a plot as an image file Click

Print a plot Click

Copy a plot to the clipboard Click

Export run data and results Click Export

Print the plate layout Select File4Print

Create slides Select File4Send to PowerPoint

Print a report Select File4Print Report

Export configurations

Data type Description File format

Plate setup files, for future Plate setup information • XLS

experiments • XLSX
For example, the well number, sample name and color,
target name, dyes, and other reaction plate contents. • TXT

Analyzed data, for further analysis QuantStudio™ format • XLS

• XLSX
• TXT

RDML (Real-Time PCR Data Markup Language) format • RDML

Used for standard curve, relative standard curve, and
comparative Ct experiments.

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 4 Set up, run, and review
standard curve experiments

■ Standard curve experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

■ Set up a standard curve experiment in the software . . . . . . . . . . . . . . . . . . . . . . . 43
■ Set up and run the PCR reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
■ Review results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Standard curve experiments

Overview Use standard curve experiments to determine absolute target quantity in samples.
In a standard curve experiment, the software performs the following tasks.
1. The software measures amplification of the target in a standard dilution series
and in test samples.
2. The software generates a standard curve using data from the standard dilution
series.
3. The software uses the standard curve to interpolate the absolute quantity of
target in the test samples.

Reaction types Multiple targets can be assayed in a standard curve experiment, but each target
requires its own standard curve.

Table 4 Reaction types for standard curve experiments

Reaction type (task) Sample description

Standard A sample that contains known or known relative quantities

of the target
• For known quantities—Quantify the target in the
standard sample using an independent method.
• For known relative quantities—Generate a relative
dilution series of the target standards.

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4 Chapter 4 Set up, run, and review standard curve experiments
Standard curve experiments

Reaction type (task) Sample description

Unknown Test sample

No-template control (NTC/ Water or buffer

Negative Control)
No amplification of the target should occur in NTC wells.

• The precision of quantification experiments improves as the number of replicate

reactions increases. Set up the number of replicates appropriate for the
experiment.
• For accurate and precise efficiency measurements, set up the standard dilution
series with at least five dilution points over a broad range of standard quantities,
4 to 6 logs (104- to 106-fold). A concentrated template, such as a plasmid or PCR
product, is best for this purpose.
A narrow range of standard quantities may be appropriate if the amount of
standard is limited, the target is in low abundance, or the target is known to fall
within a given range.

Compatible PCR Table 5 PCR options for standard curve experiments

options
Single- or multiplex PCR PCR or RT-PCR[1] Detection chemistry

Singleplex PCR TaqMan®

Multiplex 1-step RT-PCR SYBR™ Green
2-step RT-PCR
[1] RT-PCR: reverse transcription-PCR

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 Chapter 4 Set up, run, and review standard curve experiments
Set up a standard curve experiment in the software 4

Set up a standard curve experiment in the software

1. In the Home screen, create or open a template.
• In the New Experiment pane, click Create New Experiment to create a
new template.
• In the Open Existing Experiment pane, click Open to select and open an
existing template.

2. In the Properties tab, enter the template information.

3. In the Method tab, adjust the reaction volume.

For most experiments, the default run method is appropriate.

4. In the Plate tab (Quick Setup), assign plate attributes.

a. In the Plate Attributes pane, select the Passive Reference from the
dropdown list.

5. In the Plate tab (Quick Setup), define and assign well attributes.
a. Select wells in the Plate Layout or the Well Table.

b. Assign samples and targets to selected wells.

• Enter new sample and target names in the text fields.
• Select previously defined samples and targets from the dropdown lists.
Note: New sample or target names entered in the Quick Setup subtab are
automatically populated with default values for Reporter (FAM) and
Quencher (NFQ-MGB) and assigned a task ( Unknown). Edit these
values in the Advanced Setup subtab.

6. (Optional) In the Plate tab, set up standard dilutions (see “Define and set up
standard dilutions“ on page 94).

7. (Optional) In the Plate tab (Advanced Setup), assign tasks.

a. Select wells in the Plate Layout or the Well Table.

b. In the Targets table, select the checkbox of a target, then select a task from
the dropdown list.

Reaction type Task

Unknown (test sample)
No-template control

8. (Optional) In the Plate tab (Advanced Setup), define and assign biological
replicate groups (see “Define and assign biological replicate groups“ on
page 95).

In the Plate tab (Advanced Setup), ensure the Samples table contains the following
sample information:
• One sample name for each technical replicate group of an unknown sample
• (Optional) A standard sample for each target

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4 Chapter 4 Set up, run, and review standard curve experiments
Set up and run the PCR reactions

Set up and run the PCR reactions

1. Assemble the PCR reactions. Follow the manufacturer's instructions for the
reagents and follow the plate layout set up in the software (see “Prepare
reactions“ on page 21).

2. In the desktop software, open the appropriate template (EDT file).

3. Load the reaction plate into the instrument and start the run (see “Start and
monitor a run“ on page 22).

Review results
Workflow: Review View the Amplification Plot to confirm or correct
standard curve threshold and baseline settings (page 30)
experiments ▼

Assess the Standard Curve Plot (page 46)

Review data for outliers and (optional) omit wells (page 33)

(Optional) View the Multicomponent Plot to review the dye signal profile (page 36)

(Optional) View the Raw Data Plot to review the signal profile (page 37)

(Optional) Review flags in the QC Summary (page 38)

(Optional) Configure the analysis settings (page 47, page 100)

IMPORTANT! If you omit wells or configure the analysis settings, click Analyze to
reanalyze the data.

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 Chapter 4 Set up, run, and review standard curve experiments
Review results 4

Standard Curve The Standard Curve Plot displays the standard curve for samples designated as
Plot overview standards. The software calculates the quantity of an unknown target from the
standard curve.

Table 6 Results or metrics to review in the Standard Curve Plot

Results or
Description Criteria for evaluation
metrics

Slope and The amplification efficiency is calculated A slope close to –3.3 indicates optimal, 100% PCR
amplification using the slope of the regression line in amplification efficiency.
efficiency the standard curve.
Factors that affect amplification efficiency:
• Improper design of the primer and probe
• Range of standard quantities—For accurate and
precise efficiency measurements, use a broad range
of standard quantities, 5 to 6 logs (105- to 106-fold).
• Number of standard replicates—For accurate
efficiency measurements, include replicates to
decrease the effects of pipetting inaccuracies.
• PCR inhibitors—PCR inhibitors and contamination in
the reaction can reduce amplification efficiency.
• Other possible factors:
– Component and properties of the reaction mix,
such as salt content, DMSO, pH, etc.
– Inaccurate sample or reagent pipetting
– Improper analysis settings
– Incorrect plate setup

R2 value The R2 value is a measure of the closeness • A value of 1.00 indicates a perfect fit between the
(correlation of fit between the regression line and the regression line and the data points.
coefficient) individual Cq data points of the standard • An R2 value > 0.99 is desirable.
reactions.

Error The standard error of the slope of the Acceptable value is determined by the experimental
regression line in the standard curve. criteria.
The error can be used to calculate a
confidence interval (CI) for the slope and
therefore the amplification efficiency.

Ct values The threshold cycle (Ct) is the PCR cycle A Ct value > 8 and < 35 is desirable.
number at which the fluorescence level • Ct value < 8—There may be too much template in the
meets the threshold. reaction.
• Ct value > 35—There may be a low amount of target
in the reaction; for Ct values > 35, expect a higher
standard deviation.

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4 Chapter 4 Set up, run, and review standard curve experiments
Review results

View and assess You can view and assess the Standard Curve Plot in the Results tab.
the Standard If no data are displayed in the Results tab, click Analyze.
Curve Plot
1. Select Standard Curve from the dropdown list.

2. Click to configure the plot, then make the following selections:

• Target: Select the target of interest
• Plot Color: Sample, Target, or Task
• Select all wells in the Plate Layout
The Standard Curve Plot is displayed. The slope, R2 value, amplification
efficiency, and error are displayed below the plot.

3. Confirm that the slope, R2 value, amplification efficiency, and error meet the
experimental criteria.

4. Visually check that all unknown sample Cq (Ct or Crt) values fall within the
standard curve range.

5. In the Well Table, use the Group By dropdown list to confirm that the
Cq (Ct or Crt) values of all replicate samples meet the experimental criteria.

Figure 9 Example Standard Curve Plot

If the results do not meet the experimental criteria, troubleshoot using one of the
following strategies:
• Omit wells, then reanalyze. For more information, see “Omit outliers from
analysis“ on page 33.
• Repeat the experiment, adjusting the template setup and analysis settings to
improve results.
To learn more about the Standard Curve Plot, see “Standard Curve Plot overview“ on
page 45.

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 Chapter 4 Set up, run, and review standard curve experiments
Review results 4

Standard curve You can use the standard curve from another experiment and apply it to the current
settings overview experiment. The two experiments must be from the same instrument type, block type,
and run method.
To import an external standard curve, select Analysis Settings4Standard Curve
Settings, then follow the instructions on the screen.
For step-by-step instructions for adjusting the standard curve settings, see the desktop
software Help.

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 5 Set up, run, and review
relative standard curve experiments
and comparative Ct experiments

■ Relative standard curve experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

■ Comparative Ct experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
■ Relative quantitation: relative standard curve vs. comparative Ct . . . . . . . . . . . 51
■ Set up a relative standard curve experiment in the software . . . . . . . . . . . . . . . . 51
■ Set up a comparative Ct experiment in the software . . . . . . . . . . . . . . . . . . . . . . . 53
■ Set up and run the PCR reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
■ Review results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

Relative standard curve experiments

Overview Use relative standard curve experiments to determine relative target quantity in
samples.
In a relative standard curve experiment, the software performs the following tasks.
1. The software measures amplification of the target of interest and of an
endogenous control target in a standard dilution series, in a reference (calibrator)
sample, and in test samples.
The endogenous control is a target that is expressed equally in all samples;
examples of endogenous controls are β-actin, GAPDH, and 18S ribosomal RNA.
The software can algorithmically incorporate multiple endogenous control
targets in relative quantification calculations.
The reference sample is used as the basis for relative quantification results (or
1× sample). For example, in a study of drug effects on gene expression, an
untreated control is an appropriate reference sample.
2. The software generates standard curves for the target of interest and the
endogenous control using data from the corresponding standard dilution series.
3. The software uses the standard curves to interpolate the quantities of the target
of interest and the endogenous control in each sample. The target quantity in
each sample is then normalized to the sample's endogenous control quantity.
4. To determine the relative quantity of the target in test samples, the software
divides the normalized target quantity in the sample by the normalized target
quantity in the reference sample.
For a comparison of this method to the comparative Ct (DDCt) method, see “Relative
quantitation: relative standard curve vs. comparative Ct“ on page 51.

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Relative standard curve experiments 5

Reaction types Relative standard curve experiments include the following reaction types for the
endogenous control target and each target of interest.

Table 7 Reaction types for relative standard curve experiments

Reaction type (task) Sample description

Standard A sample that contains known or known relative quantities

of the target
• For known quantities—Quantify the target in the
standard sample using an independent method.
• For known relative quantities—Generate a relative
dilution series of the target standards.

Reference sample[1] The sample that is used as the basis for relative
quantification results

Unknown Test or reference sample

No-template control (NTC/ Water or buffer

Negative Control)
No amplification of the target should occur in NTC wells.
[1] To identify a sample as a reference sample, review the relative quantification settings.

• The precision of quantification experiments improves as the number of replicate

reactions increases. Set up the number of replicates appropriate for the
experiment.
• For accurate and precise efficiency measurements, set up the standard dilution
series with at least five dilution points over a broad range of standard quantities,
4 to 6 logs (104- to 106-fold). A concentrated template, such as a plasmid or PCR
product, is best for this purpose.
A narrow range of standard quantities may be appropriate if the amount of
standard is limited, the target is in low abundance, or the target is known to fall
within a given range.

Compatible PCR Table 8 PCR options for relative standard curve experiments
options
Single- or multiplex PCR PCR or RT-PCR[1] Detection chemistry

Singleplex PCR TaqMan®

Multiplex 1-step RT-PCR SYBR™ Green
2-step RT-PCR
[1] RT-PCR: reverse transcription PCR.

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5 Chapter 5 Set up, run, and review relative standard curve experiments and comparative Ct experiments
Comparative Ct experiments

Comparative Ct experiments
Overview Use comparative Ct (DDCt) experiments to determine relative target quantity in
samples.
In a comparative Ct experiment, the software performs the following tasks.
1. The software measures amplification of the target of interest and of an
endogenous control target in a reference (calibrator) sample and in test samples.
The endogenous control is a target that is expressed equally in all samples;
examples of endogenous controls are β-actin, GAPDH, and 18S ribosomal RNA.
The software can algorithmically incorporate multiple endogenous control
targets in relative quantification calculations.
The reference sample is used as the basis for relative quantification results (or
1× sample). For example, in a study of drug effects on gene expression, an
untreated control is an appropriate reference sample.
2. The measurements for the target of interest are normalized to the endogenous
control.
3. To determine the relative quantity of the target in test samples, the software
compares the normalized ΔCq (ΔCt or ΔCrt) for the sample to the normalized
ΔCq (ΔCt or ΔCrt) for the reference sample.
For a comparison of this method to the relative standard curve method, see “Relative
quantitation: relative standard curve vs. comparative Ct“ on page 51.

Reaction types Comparative Ct experiments include the following reaction types for the endogenous
control target and each target of interest.

Table 9 Reaction types for comparative Ct experiments

Reaction type (task) Sample description

Reference sample[1] The sample that is used as the basis for relative
quantification results

Unknown Test or reference sample

No-template control (NTC/ Water or buffer

Negative Control)
No amplification of the target should occur in NTC wells.
[1] To identify a sample as a reference sample, review the relative quantification settings.

The precision of quantification experiments improves as the number of replicate

reactions increases. Set up the number of replicates appropriate for the experiment.

Compatible PCR Table 10 PCR options for comparative Ct experiments

options
Single- or multiplex PCR PCR or RT-PCR[1] Detection chemistry

Singleplex PCR TaqMan®

Multiplex 1-step RT-PCR SYBR™ Green
2-step RT-PCR
[1] RT-PCR: reverse transcription PCR

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Relative quantitation: relative standard curve vs. comparative Ct 5

Relative quantitation: relative standard curve vs. comparative Ct

Use either relative standard curve experiments or comparative Ct experiments to
determine the relative quantity of a target of interest in a test sample relative to a
reference sample. Relative quantitation experiments are commonly used for the
following applications.
• Comparison of expression levels of a gene in different tissues.
• Comparison of expression levels of a gene in a treated sample vs. an untreated
sample.
• Comparison of expression levels of a gene of interest in different genetic
backgrounds.
• Analysis of the gene expression changes over time under specific treatment
conditions.

Table 11 Comparison of relative standard curve experiments and comparative Ct experiments

Characteristic Relative standard curve Comparative Ct

Typical use Best for assays that have suboptimal PCR Best for high-throughput measurements of
efficiency. relative gene expression of many genes in many
samples.

Advantage Requires the least amount of validation • Relative levels of target in samples can be
because the PCR efficiencies of the target and determined without the use of a standard
endogenous control do not need to be curve, if the PCR efficiencies of the target
equivalent. and endogenous control are relatively
equivalent.
• Reduced reagent usage.
• More space available in the reaction plate.

Limitation A standard curve must be constructed for each • Suboptimal (low PCR efficiency) assays
target, which requires more reagents and more may produce inaccurate results.
space in the reaction plate. • Before you use the comparative Ct method,
we recommend that you determine that
the PCR efficiencies for the target assay
and the endogenous control assay are
approximately equal.

Set up a relative standard curve experiment in the software

1. In the Home screen, create or open a template.
• In the New Experiment pane, click Create New Experiment to create a
new template.
• In the Open Existing Experiment pane, click Open to select and open an
existing template.

2. In the Properties tab, enter the template information.

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5 Chapter 5 Set up, run, and review relative standard curve experiments and comparative Ct experiments
Set up a relative standard curve experiment in the software

3. In the Method tab, adjust the reaction volume.

For most experiments, the default run method is appropriate.

4. In the Plate tab (Quick Setup), assign plate attributes.

a. In the Plate Attributes pane, select a Passive Reference, Reference Sample,
and Endogenous Control from the dropdown lists.

5. In the Plate tab (Quick Setup), define and assign well attributes.
a. Select wells in the Plate Layout or the Well Table.

b. Assign samples and targets to selected wells.

• Enter new sample and target names in the text fields.
• Select previously defined samples and targets from the dropdown lists.
Note: New sample or target names entered in the Quick Setup subtab are
automatically populated with default values for Reporter (FAM) and
Quencher (NFQ-MGB) and assigned a task ( Unknown). Edit these
values in the Advanced Setup subtab.

6. In the Plate tab, set up standard dilutions (see “Define and set up standard
dilutions“ on page 94).

7. (Optional) In the Plate tab (Advanced Setup), assign tasks.

a. Select wells in the Plate Layout or the Well Table.

b. In the Targets table, select the checkbox of a target, then select a task from
the dropdown list.

Reaction type Task

Unknown (test sample)
No-template control

8. (Optional) In the Plate tab (Advanced Setup), define and assign biological
replicate groups (see “Define and assign biological replicate groups“ on
page 95).

The targets of interest and the endogenous control target should each have wells that
are assigned with standard dilutions, unknown, and no-template-control tasks, and
corresponding samples.
In the Plate tab (Advanced Setup), ensure the Samples table contains the following
samples.
• Unknown samples
• Reference sample
• (Optional) For a dilution of target standards, each dilution step for each
endogenous control target and target of interest has its own sample name.

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Set up a comparative Ct experiment in the software 5

Set up a comparative Ct experiment in the software

1. In the Home screen, create or open a template.
• In the New Experiment pane, click Create New Experiment to create a
new template.
• In the Open Existing Experiment pane, click Open to select and open an
existing template.

2. In the Properties tab, enter the template information.

3. In the Method tab, adjust the reaction volume.

For most experiments, the default run method is appropriate.

4. In the Plate tab (Quick Setup), assign plate attributes.

a. In the Plate Attributes pane, select a Passive Reference, Reference Sample,
and Endogenous Control from the dropdown lists.

5. In the Plate tab (Quick Setup), define and assign well attributes.
a. Select wells in the Plate Layout or the Well Table.

b. Assign samples and targets to selected wells.

• Enter new sample and target names in the text fields.
• Select previously defined samples and targets from the dropdown lists.
Note: New sample or target names entered in the Quick Setup subtab are
automatically populated with default values for Reporter (FAM) and
Quencher (NFQ-MGB) and assigned a task ( Unknown). Edit these
values in the Advanced Setup subtab.

6. (Optional) In the Plate tab (Advanced Setup), assign tasks.

a. Select wells in the Plate Layout or the Well Table.

b. In the Targets table, select the checkbox of a target, then select a task from
the dropdown list.

Reaction type Task

Unknown (test sample)
No-template control

7. (Optional) In the Plate tab (Advanced Setup), define and assign biological
replicate groups (see “Define and assign biological replicate groups“ on
page 95).

The targets of interest and the endogenous control target should each have wells
assigned with unknown and no-template-control tasks, and corresponding samples.
In the Plate tab (Advanced Setup), ensure the Samples table contains:
• Unknown samples
• Reference sample

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5 Chapter 5 Set up, run, and review relative standard curve experiments and comparative Ct experiments
Set up and run the PCR reactions

Set up and run the PCR reactions

1. Assemble the PCR reactions. Follow the manufacturer's instructions for the
reagents and follow the plate layout set up in the software (see “Prepare
reactions“ on page 21).

2. In the desktop software, open the appropriate template (EDT file).

3. Load the reaction plate into the instrument and start the run (see “Start and
monitor a run“ on page 22).

Review results
Workflow: Review Relative standard curve experiments Comparative Ct experiments
relative standard
curve and View the Amplification Plot to confirm or correct
threshold and baseline settings (page 30)
comparative Ct
experiments ▼ ▼

Assess the Standard Curve Plot (page 46) —

▼ ▼

Review data for outliers and (optional) omit wells (page 33)

Assess the Gene Expression Plot (page 57)

(Optional) View the Endogenous Control Profile using the QC Plot (page 58)

(Optional) View the Multicomponent Plot to review the dye signal profile (page 36)

(Optional) View the Raw Data Plot to review the signal profile (page 37)

(Optional) Review flags in the QC Summary (page 38)

(Optional) Configure the analysis settings (page 59, page 100)

IMPORTANT! If you omit wells or configure the analysis settings, click Analyze to
reanalyze the data.

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Review results 5

View and assess This section only applies to relative standard curve experiments.
the Standard
Curve Plot Standard Curve Plot overview
The Standard Curve Plot displays the standard curve for samples designated as
standards. The software calculates the quantity of an unknown target from the
standard curve.

Table 12 Results or metrics to review in the Standard Curve Plot

Results or
Description Criteria for evaluation
metrics

Slope and The amplification efficiency is calculated A slope close to –3.3 indicates optimal, 100% PCR
amplification using the slope of the regression line in amplification efficiency.
efficiency the standard curve.
Factors that affect amplification efficiency:
• Improper design of the primer and probe
• Range of standard quantities—For accurate and
precise efficiency measurements, use a broad range
of standard quantities, 5 to 6 logs (105- to 106-fold).
• Number of standard replicates—For accurate
efficiency measurements, include replicates to
decrease the effects of pipetting inaccuracies.
• PCR inhibitors—PCR inhibitors and contamination in
the reaction can reduce amplification efficiency.
• Other possible factors:
– Component and properties of the reaction mix,
such as salt content, DMSO, pH, etc.
– Inaccurate sample or reagent pipetting
– Improper analysis settings
– Incorrect plate setup

R2 value The R2 value is a measure of the closeness • A value of 1.00 indicates a perfect fit between the
(correlation of fit between the regression line and the regression line and the data points.
coefficient) individual Cq data points of the standard • An R2 value > 0.99 is desirable.
reactions.

Error The standard error of the slope of the Acceptable value is determined by the experimental
regression line in the standard curve. criteria.
The error can be used to calculate a
confidence interval (CI) for the slope and
therefore the amplification efficiency.

Ct values The threshold cycle (Ct) is the PCR cycle A Ct value > 8 and < 35 is desirable.
number at which the fluorescence level • Ct value < 8—There may be too much template in the
meets the threshold. reaction.
• Ct value > 35—There may be a low amount of target
in the reaction; for Ct values > 35, expect a higher
standard deviation.

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5 Chapter 5 Set up, run, and review relative standard curve experiments and comparative Ct experiments
Review results

View and assess the Standard Curve Plot

You can view and assess the Standard Curve Plot in the Results tab.
If no data are displayed in the Results tab, click Analyze.

1. Select Standard Curve from the dropdown list.

2. Click to configure the plot, then make the following selections:

• Target: Select the target of interest
• Plot Color: Sample, Target, or Task
• Select all wells in the Plate Layout
The Standard Curve Plot is displayed. The slope, R2 value, amplification
efficiency, and error are displayed below the plot.

3. Confirm that the slope, R2 value, amplification efficiency, and error meet the
experimental criteria.

4. Visually check that all unknown sample Cq (Ct or Crt) values fall within the
standard curve range.

5. In the Well Table, use the Group By dropdown list to confirm that the
Cq (Ct or Crt) values of all replicate samples meet the experimental criteria.

Figure 10 Example Standard Curve Plot

If the results do not meet the experimental criteria, troubleshoot using one of the
following strategies:
• Omit wells, then reanalyze. For more information, see “Omit outliers from
analysis“ on page 33.
• Repeat the experiment, adjusting the template setup and analysis settings to
improve results.
To learn more about the Standard Curve Plot, see “Standard Curve Plot overview“ on
page 45.

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Review results 5

Standard curve settings overview

You can use the standard curve from another experiment and apply it to the current
experiment. The two experiments must be from the same instrument type, block type,
and run method.
To import an external standard curve, select Analysis Settings4Standard Curve
Settings, then follow the instructions on the screen.
For step-by-step instructions for adjusting the standard curve settings, see the desktop
software Help.

Gene Expression The Gene Expression Plot displays the results of relative quantification calculations
Plot overview for relative standard curve and comparative Ct experiments.
Review the Gene Expression Plot to evaluate the fold change in expression level of the
targets of interest in the test samples relative to the reference sample.
There are two plots available, depending on the experimental focus. Each plot can be
viewed on a linear, log10, Ln, and log2 scale.

Table 13 Gene Expression plots

Plot type Description

RQ vs. Target Groups the relative quantification (RQ) values by target.

Each sample is plotted for each target.

RQ vs. Sample Groups the relative quantification (RQ) values by sample.

Each target is plotted for each sample.

Figure 11 Example Gene Expression Plot

In this example, there is one target of interest, and the reference sample (calibrator) is sample
800.

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Review results

QC Plot overview The QC Plot is a visual display of the Cq (Ct or Crt) levels of potential endogenous
control targets across all samples (Endogenous Control Profile).
Use the QC Plot to help choose the best endogenous control for an experiment. Select
the target with a quantity (indicated by Cq (Ct or Crt) value) that does not change
under experimental conditions.
All targets can be displayed in the QC Plot. You can view up to four potential
endogenous controls at a time.

View and assess the QC Plot

You can view and assess the QC Plot in the Results tab.
If no data are displayed, click Analyze.

1. In the Results tab, select QC Plot from the dropdown list.

The Endogenous Control Profile is displayed.

2. In the right pane, select the targets to display, then select the color and shape
from the dropdown lists.

3. (Optional) In the View Replicate Results Table tab, select the samples to omit
from analysis.

4. (Optional) To change the endogenous controls used for analysis, select

Analysis Settings4Relative Quantification Settings (see “Relative
quantification settings overview“ on page 59).

5. Click Analyze to see the result of the adjustments.

Figure 12 Example QC Plot

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Review results 5

Relative In the Results tab, select Analysis Settings4Relative Quantification Settings to

quantification configure the following parameters:
settings overview Parameter Description

Analysis Type Select Multiplex or Singleplex analysis.

References Set the reference sample, or set a biological replicate

group as the reference sample.

Endogenous Controls Change the endogenous control, or select multiple

endogenous controls.

Efficiency Set the amplification efficiency for a target.

(Comparative Ct The amplification efficiency for each target is calculated
experiments only) from the standard dilution series in relative standard curve
experiments.

Outlier Rejection Outliers with ΔCq (ΔCt or ΔCrt) values less than or equal to
the entered value are rejected.
(Multiplex reactions only)

RQ Min/Max Calculations Determines the algorithm used to calculate the relative

quantification minimum and maximum values (error bars).
• Confidence Level—Select to calculate the RQ
minimum and maximum values based on the selected
confidence level.
• Standard Deviations—Select to calculate the RQ
minimum and maximum values based on the selected
number of standard deviations.

For step-by-step instructions for adjusting the relative quantification settings, see the
desktop software Help.

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 6 Set up, run, and review
genotyping experiments

■ Genotyping experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
■ Set up a genotyping experiment in the software . . . . . . . . . . . . . . . . . . . . . . . . . . 61
■ Set up and run the PCR reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
■ Review results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Genotyping experiments
Overview Use genotyping experiments to detect single nucleotide polymorphism (SNP) variants
of a target nucleic acid sequence.
Genotyping experiments use preformulated TaqMan® SNP Genotyping Assays that
include the following components:
• Two sequence-specific primers for amplification of sequences containing the SNP
of interest
• Two allele-specific TaqMan® probes for Allele 1 and Allele 2
In a genotyping experiment, the software performs the following tasks.
1. The software normalizes the fluorescence of the reporter dyes to the fluorescence
of the passive reference dye in each well.
2. The software plots the normalized reporter dye signal of each sample well on an
Allelic Discrimination Plot, which contrasts the reporter dye intensities of the
allele-specific probes.
3. The software algorithmically clusters the sample data, and assigns a genotype
call to the samples of each cluster according to its position on the plot.

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 Chapter 6 Set up, run, and review genotyping experiments
Set up a genotyping experiment in the software 6

Reaction types Table 14 Reaction types for genotyping experiments

Reaction type (task) Sample description

Unknown Test sample

No-template control Water or buffer

No amplification of the target should occur in NTC wells.

Allele control (1/1) Control sample that is homozygous for allele 1

Allele control (1/2) Control sample that is heterozygous allele 1/allele 2

Allele control (2/2) Control sample that is homozygous for allele 2

Allele controls are optional but recommended. Including allele controls helps to
improve the clustering algorithm, particularly in situations where a limited number of
samples are run.
In genotyping experiments, the software makes calls for individual wells. Running 3
or more replicates of each reaction can help identify outlier wells that may be present.

Compatible PCR Table 15 PCR options for genotyping experiments

options
Single- or multiplex PCR PCR or RT-PCR[1] Detection chemistry

Multiplex[2] PCR TaqMan®

[1] RT-PCR: reverse transcription PCR
[2] Each SNP genotyping assay is a multiplex assay with a probe for each allele. Multiple SNP assays can be
performed in a single well.

Genotyping calls are based either on end-point data (data collected outside of any
PCR cycling stage) or on real-time data (data collected during a PCR cycling stage).
For detailed information, see the analysis settings section of this guide.
We recommend collecting real-time amplification data during the PCR stage, for
troubleshooting purposes.

Set up a genotyping experiment in the software

1. In the Home screen, create or open a template.
• In the New Experiment pane, click Create New Experiment to create a
new template.
• In the Open Existing Experiment pane, click Open to select and open an
existing template.

2. In the Properties tab, enter the template information.

3. In the Method tab, adjust the reaction volume.

For most experiments, the default run method is appropriate.

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6 Chapter 6 Set up, run, and review genotyping experiments
Set up a genotyping experiment in the software

4. In the Plate tab (Quick Setup), assign plate attributes.

a. In the Plate Attributes pane, select the Passive Reference from the
dropdown list.

5. In the Plate tab (Quick Setup), define and assign well attributes.
a. Select wells in the Plate Layout or the Well Table.

b. Assign samples and SNP assays to selected wells.

• Enter new sample and SNP assay names in the text fields.
• Select previously defined samples and SNP assays from the dropdown
lists.
Note: New sample or SNP assay names entered in the Quick Setup subtab
are automatically populated with the following default values:

Allele 1 Reporter Quencher Allele 2 Reporter Quencher Task

Allele 1 VIC NFQ- Allele 2 FAM NFQ-
MGB MGB Unknown

Edit these values in the Advanced Setup subtab.

6. (Optional) In the Plate tab (Advanced Setup), assign tasks.

a. Select wells in the Plate Layout or the Well Table.

b. In the SNP Assays table, select the checkbox of a SNP assay, then select a
task from the dropdown list.

Reaction type Task

Unknown (test sample)
No-template control
Allele control (1/1)[1]
Allele control (1/2)[1]
Allele control (2/2)[1]
[1] Optional but recommended

In the Plate tab (Advanced Setup), ensure the Samples table contains the following
samples:
• Unknown samples
• (Optional) Allele control samples

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Set up and run the PCR reactions 6

Set up and run the PCR reactions

1. Assemble the PCR reactions. Follow the manufacturer's instructions for the
reagents and follow the plate layout set up in the software (see “Prepare
reactions“ on page 21).

2. In the desktop software, open the appropriate template (EDT file).

3. Load the reaction plate into the instrument and start the run (see “Start and
monitor a run“ on page 22).

Review results
Workflow: Review Assess the Allelic Discrimination Plot (page 64)
genotyping
▼
experiments
(Optional) View the Amplification Plot (page 30)

Review data for outliers and (optional) omit wells (page 33)

(Optional) View the Multicomponent Plot to review the dye signal profile (page 36)

(Optional) View the Raw Data Plot to review the signal profile (page 37)

(Optional) Review flags in the QC Summary (page 38)

(Optional) Configure the analysis settings (page 66, page 100)

IMPORTANT! If you omit wells or configure the analysis settings, click Analyze to
reanalyze the data.

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6 Chapter 6 Set up, run, and review genotyping experiments
Review results

Allelic The Allelic Discrimination Plot contrasts the Rn or the ΔRn of the reporter dyes for the
Discrimination allele-specific probes of the SNP assay. It is an intermediary step in the software
algorithm for genotyping calls.
Plot overview
Data points tend to cluster along the horizontal axis (Allele 1), vertical axis (Allele 2),
or diagonal (Allele 1/Allele 2).

Table 16 Data clusters in the Allelic Discrimination Plot

A substantial increase in... Clusters along... Indicates...

Fluorescence of Horizontal axis Homozygosity for Allele 1

VIC™ dye-labeled probe only

Fluorescence of Vertical axis Homozygosity for Allele 2

FAM™ dye-labeled probe only

Fluorescence of both Diagonal Heterozygosity for

VIC™ and FAM™ dye-labeled probes Allele 1 – Allele 2

Review the allelic discrimination plot to assess data clusters.

• Confirm that clustering of control samples is as expected.
• Visually assess clusters for the three possible genotypes.
Note: The desktop software clustering algorithm does not call genotypes if all the
samples are one genotype (form one cluster).

View and assess You can view and assess the Allelic Discrimination Plot in the Results tab.
the Allelic If no data are displayed in the Results tab, click Analyze.
Discrimination
Plot 1. In the Results tab, select Allelic Discrimination Plot from the dropdown list.

2. Click to configure the plot, then make the following selections:

• SNP Assay: select the assay of interest
• Plot Type: Cartesian or Polar
The Allelic Discrimination Plot is displayed for the selected SNP assay.
Note: Initially, all points in the plot are cyan because all of the wells in the
Plate Layout are selected. Click anywhere in the plot or Plate Layout to
deselect all wells. The data points in the plot change to the call colors.

3. Confirm that control data clusters as expected.

a. In the Well Table or Plate Layout, select the wells containing a
control to highlight the corresponding data points in the plot.

b. Check that the data points for each genotype control cluster along the
expected axis of the plot.

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 Chapter 6 Set up, run, and review genotyping experiments
Review results 6

4. Select the cluster at the bottom-left corner of the plot, then confirm that only the
negative control wells are selected in the Plate Layout or Well Table.
Samples can unexpectedly cluster with the negative controls for one of the
following reasons.
• Samples contain no DNA.
• Samples contain PCR inhibitors.
• Samples are homozygous for a sequence deletion.

5. Review the other clusters in the plot.

a. Click–drag a box around a cluster to select the associated wells.

b. Confirm that the expected wells are selected in the Plate Layout or
Well Table.

6. Look for outliers outside the three genotype clusters.

Figure 13 Example Allelic Discrimination Plot

To confirm results, retest outliers and samples with no amplification (cluster with
negative controls).

Perform manual You can perform manual calls in the Results tab.
calls 1. In the Results tab, select Allelic Discrimination Plot from the dropdown list.

2. If the data are not analyzed, click Analyze.

3. (For multiple assays only) Click , then select a SNP assay from the dropdown
list.

4. In the Allelic Discrimination Plot, use the lasso tool to select the samples to be
manually called.

5. Click , then select the allele call from the Apply Call dropdown list.

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 6 Chapter 6 Set up, run, and review genotyping experiments
Review results

6. Click Analyze.

IMPORTANT! To maintain manual calls after reanalysis, select Analysis

Settings4Call Settings, then deselect Default Settings and select Keep Manual
Calls from Previous Analysis.

Note: To remove manual calls, select Analysis Settings4Call Settings,

deselect Keep Manual Calls from Previous Analysis, then reanalyze.

Call settings In the Results tab, select Analysis Settings4Call Settings to edit the following
overview settings:
(genotyping) • Data analysis settings
• Default call settings for SNP assays without custom call settings
• Custom call settings for individual SNP assays

Table 17 Options for data analysis settings (genotyping experiments)

Data analysis setting Description

Analyze Data from Post-PCR Read Only Only post-PCR read data is used to determine calls.

Analyze Data from Pre-PCR Read and Post-PCR Read[1] The pre-PCR read is subtracted from the post-PCR read
to determine calls.

Analyze Real-Time Rn Data[2,3] The normalized reporter data (Rn) from the user-
selected cycle of the cycling stage is used to determine
calls.

Analyze Real-Time Rn - Median (Rna to Rnb)[2,4,3] A quick baseline-subtracted Rn from the user-selected
cycle of the cycling stage is used to determine calls. The
quick baseline-subtracted Rn is the Rn minus the median
value of the baseline region.
The median subtraction provides improved data accuracy.

Analyze Real-Time dRn Data[2,3] The regular ΔRn (dRn) from the user-selected cycle of
the cycling stage is used to determine calls. The ΔRn is
calculated by subtracting the best-fit line through the
baseline region.
This method is better if the baselines are not flat.
[1] The run method must include a pre-read stage.
[2] Data collection must be on during the PCR stage.
[3] Analysis is not restricted to the last cycle; adjust the analysis cycle using the Reveal Traces feature while viewing the Allelic Discrimination Plot.
[4] Rna to Rnb refers to all the cycles from the Start Cycle Number to the End Cycle Number of the baseline region.

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 Chapter 6 Set up, run, and review genotyping experiments
Review results 6

Table 18 Options for call settings (genotyping experiments)

Call setting Description

Autocaller Enabled The autocaller algorithm is used to make genotype calls.

Keep Manual Calls from Previous Analysis If autocaller is enabled, maintains manual calls after reanalysis.

Quality Value The Quality Value is a proprietary estimation of the likelihood that
a genotyping call is correct (associated with the correct cluster).
If the Quality Value is less than the setting, the call is
undetermined.

For step-by-step instructions for adjusting the call settings, see the desktop software
Help.

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 7 Set up, run, and review
presence/absence experiments

■ Presence/absence experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
■ Set up the presence/absence experiment in the software . . . . . . . . . . . . . . . . . . . 69
■ Set up and run the PCR reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
■ Review results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

Presence/absence experiments
Overview Use presence/absence experiments to determine the presence or absence of a target
nucleic acid sequence in a sample.
The software calls the target present or absent based on an algorithmically determined
call threshold. (The call threshold is different from the Ct threshold; the Ct threshold is
not used to make calls.)

Reaction types Presence/absence reaction types depend on whether the experiment is set up with or
without an internal positive control (IPC).
• Presence/absence experiments with IPC (recommended) are multiplex assays
for the target of interest and the IPC target. The IPC is used to confirm that a
negative result for the target of interest is not caused by a failed PCR.

Table 19 Reaction types for presence/absence experiments with IPC

Reaction type (task) Sample description

Unknown Test sample
and
IPC template
Negative control Water or buffer
and
IPC template
No amplification control Water or buffer plus a blocking agent
(NAC; blocked IPC)[1] and
IPC template; amplification prevented by blocking agent
[1] Minimum of two replicates is required for this control.

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 Chapter 7 Set up, run, and review presence/absence experiments
Set up the presence/absence experiment in the software 7

• Presence/absence experiments without IPC are singleplex reactions.

Table 20 Reaction types for presence/absence experiments without IPC

Reaction type (task) Sample description

Unknown Test sample
Negative control Water or buffer

The software makes calls for individual wells. Running three or more replicates of
each reaction can help identify outlier wells that may be present.

Compatible PCR Table 21 PCR options for presence/absence experiments

options
Single- or multiplex PCR PCR or RT-PCR[1] Detection chemistry

Singleplex (without IPC) PCR TaqMan®

Multiplex (with IPC) 1-step RT-PCR
2-step RT-PCR
[1] RT-PCR: reverse transcription-PCR

Presence/absence calls are based on end-point data (data collected after the PCR
stage).
• The data collected is the normalized intensity of the reporter dye, or Rn.
• If end-point experiments include pre-PCR data points, the software calculates the
delta Rn (ΔRn) value according to the following formula:
ΔRn = Rn (post-PCR read) – Rn (pre-PCR read) , where Rn = normalized readings.
We recommend collecting real-time amplification data during the PCR stage, for
troubleshooting purposes.

Set up the presence/absence experiment in the software

1. In the Home screen, create or open a template.
• In the New Experiment pane, click Create New Experiment to create a
new template.
• In the Open Existing Experiment pane, click Open to select and open an
existing template.

2. In the Properties tab, enter the template information.

3. In the Method tab, adjust the reaction volume.

For most experiments, the default run method is appropriate.

4. In the Plate tab (Quick Setup), assign plate attributes.

a. In the Plate Attributes pane, select the Passive Reference from the
dropdown list.

5. In the Plate tab (Quick Setup), define and assign well attributes.
a. Select wells in the Plate Layout or the Well Table.

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7 Chapter 7 Set up, run, and review presence/absence experiments
Set up and run the PCR reactions

b. Assign samples and targets to selected wells.

• Enter new sample and target names in the text fields.
• Select previously defined samples and targets from the dropdown lists.
Note: New sample or target names entered in the Quick Setup subtab are
automatically populated with default values for Reporter (FAM) and
Quencher (NFQ-MGB) and assigned a task ( Unknown). Edit these
values in the Advanced Setup subtab.

6. (Optional) In the Plate tab (Advanced Setup), assign tasks.

a. Select wells in the Plate Layout or the Well Table.

b. In the Targets table, select the checkbox of a target, then select a task from
the dropdown list.

Reaction type Target Task

Target of interest
Unknown (test sample)
IPC
Target of interest
Negative control
IPC
Target of interest
NAC (blocked IPC)
IPC

Set up and run the PCR reactions

1. Assemble the PCR reactions. Follow the manufacturer's instructions for the
reagents and follow the plate layout set up in the software (see “Prepare
reactions“ on page 21).

2. In the desktop software, open the appropriate template (EDT file).

3. Load the reaction plate into the instrument and start the run (see “Start and
monitor a run“ on page 22).

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 Chapter 7 Set up, run, and review presence/absence experiments
Review results 7

Review results
Workflow: Review Assess the Presence/Absence Plot (page 72)
presence/absence
▼
experiments
(Optional) View the Amplification Plot (page 30)

Review data for outliers and (optional) omit wells (page 33)

(Optional) View the Multicomponent Plot to review the dye signal profile (page 36)

(Optional) View the Raw Data Plot to review the signal profile (page 37)

(Optional) Review flags in the QC Summary (page 38)

(Optional) Configure the analysis settings (page 73, page 100)

IMPORTANT! If you omit wells or configure the analysis settings, click Analyze to
reanalyze the data.

Presence/Absence The Presence/Absence Plot displays the intensity of the fluorescence for each well.
Plot overview Review the Presence/Absence Plot to confirm that amplification in the control wells is
as expected and to review the calls for the unknown samples.

Table 22 Expected results for control reactions

Reaction type Target Result Call

Negative control IPC Amplification IPC Succeeded

Target of interest[1] No amplification Negative control

NAC (blocked IPC) IPC[2] No amplification Blocked IPC Control

Target of interest No amplification Negative control

[1] The target threshold is calculated from the negative control reactions.
[2] The IPC threshold is calculated from the NAC reactions.

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7 Chapter 7 Set up, run, and review presence/absence experiments
Review results

Table 23 Criteria for calls in unknown reactions

Target signal IPC Signal Call

Above the target threshold Above or below the IPC threshold Presence

Below the target threshold Above the IPC threshold Absence

Below the target threshold Below the IPC threshold Unconfirmed

View and assess You can view and assess the Presence/Absence Plot in the Results tab.
the If no data are displayed in the Results tab, click Analyze.
Presence/Absence
Plot 1. In the Results tab, select Presence/Absence Plot from the dropdown list.

2. Click to configure the plot, making the following selections:

• Target Reporter: target defined for the target of interest
• Control Reporter: target defined for the IPC
• For the initial review of the Presence/Absence Plot, select the following
options:
– Show Calls: All Calls
– Show IPC
– Show Controls
The Presence/Absence Plot is displayed for data points selected in the plot
settings. The data points for selected wells in the Plate Layout or Well
Table are highlighted in the plot (see Figure 14).

3. Confirm that amplification in the negative and blocked IPC control wells is as
expected. Use one of the following options:
• Select control wells in the Plate Layout or Well Table, then confirm the
location of the data points in the Presence/Absence Plot.
• In the Well Table, select Group By4Task, then examine the wells with the
Blocked IPC ( ) and NTC ( ) tasks. The Cq (Ct or Crt) values should be
Undetermined.
• View the amplification plots for the negative controls (see Figure 15 and
“Optimize display of negative controls in the Amplification Plot“ on page 34).

4. In the Presence/Absence Plot, view the signal intensity and calls for the
unknown samples.
Use the plot settings (click ) to filter out the IPC results and control wells, or to
select only one type of call.

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 Chapter 7 Set up, run, and review presence/absence experiments
Review results 7

Figure 14 Example Presence/Absence Plot

The IPC results are not displayed in this example.

Figure 15 Example Amplification Plot for negative control and blocked IPC
Amplification of the IPC target (blue lines) is seen in the negative control wells but not the
blocked IPC (NAC) wells. No amplification of the target of interest (red lines) is seen in either
negative control or blocked IPC wells.

Call settings Use the Call Settings tab to edit the following settings:
overview • Data analysis settings
(presence / • Default call settings for assays without custom call settings
absence) • Custom call settings for individual assays

Table 24 Options for data analysis settings (presence/absence experiments)

Data analysis setting Description

Analyze Data from Post‑PCR Read Only Only post-PCR read data is used to
determine calls.

Analyze Data from Pre‑PCR Read and The pre-PCR read is subtracted from the
Post‑PCR Read post-PCR read to determine calls.

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7 Chapter 7 Set up, run, and review presence/absence experiments
Review results

Table 25 Options for call settings (presence/absence experiments)

Call setting Description

Confidence Value What confidence value is used to determine the target and
IPC call thresholds.
• A lower confidence value or more controls typically
results in a lower calculated threshold.
• A higher confidence value or fewer controls typically
results in a higher calculated threshold.

For step-by-step instructions for adjusting the call settings, see the desktop software
Help.

74 QuantStudio™ Design and Analysis Desktop Software User Guide

 8 Set up, run, and review
melt curve experiments

■ Melt curve experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

■ Set up a melt curve experiment in the software . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
■ Set up melt curve reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
■ Review results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Melt curve experiments

Overview Use melt curve experiments to determine the melting temperature (Tm) of the
amplification products of a PCR that used intercalating dyes.
Melting temperature (Tm) is the temperature at which 50% of the DNA is double-
stranded and 50% is dissociated into single-stranded DNA. The melt curve of a single
amplification product displays a single peak at the product's Tm. Multiple peaks in a
melt curve experiment indicate additional amplification products, usually from non-
specific amplification or formation of primer-dimers.
In the software, melt curve analysis is included in the default run method for any
experiment type that uses intercalating dyes.
1. The software plots a melt curve based on the fluorescence of the dye with respect
to change in temperature.
2. Using the melt curve, the software calculates the melting temperature (Tm).

Reaction types Table 26 Reaction types for melt curve experiments

Reaction type (task) Sample description

Unknown Previously run PCR reactions that used intercalating dyes

No-template control (NTC/ Previously run PCR reactions that used water or buffer
Negative Control)
Note: No DNA should be present in NTC wells.

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8 Chapter 8 Set up, run, and review melt curve experiments
Set up a melt curve experiment in the software

Set up a melt curve experiment in the software

1. In the Home screen, create or open a template.
• In the New Experiment pane, click Create New Experiment to create a
new template.
• In the Open Existing Experiment pane, click Open to select and open an
existing template.

2. In the Properties tab, enter the template information.

3. In the Method tab, adjust the reaction volume.

(Optional) Edit the ramp increment in the melt curve (see “Edit the ramp
increment for the melt curve dissociation step“ on page 91).

4. In the Plate tab (Quick Setup), assign plate attributes.

a. In the Plate Attributes pane, select the Passive Reference from the
dropdown list.

5. In the Plate tab (Quick Setup), define and assign well attributes.
a. Select wells in the Plate Layout or the Well Table.

b. Assign samples and targets to selected wells.

• Enter new sample and target names in the text fields.
• Select previously defined samples and targets from the dropdown lists.
Note: New sample or target names entered in the Quick Setup subtab are
automatically populated with default values for Reporter (FAM) and
Quencher (NFQ-MGB) and assigned a task ( Unknown). Edit these
values in the Advanced Setup subtab.

6. (Optional) In the Plate tab (Advanced Setup), assign tasks.

a. Select wells in the Plate Layout or the Well Table.

b. In the Targets table, select the checkbox of a target, then select a task from
the dropdown list.

Reaction type Task

Unknown (test sample)
No-template control

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 Chapter 8 Set up, run, and review melt curve experiments
Set up melt curve reactions 8

Set up melt curve reactions

Melt curve experiments are performed using previously amplified PCR products,
usually at the end of the PCR run method. You can also use a plate from an
intercalating dyes-based PCR run on another instrument.

Review results
Workflow: Review Assess the Melt Curve Plot (page 78)
melt curve
▼
experiments
(Optional) View the Multicomponent Plot to review the dye signal profile (page 36)

(Optional) View the Raw Data Plot to review the signal profile (page 37)

(Optional) Review flags in the QC Summary (page 38)

(Optional) Configure the analysis settings (page 78, page 100)

IMPORTANT! If you omit wells or configure the analysis settings, click Analyze to
reanalyze the data.

Melt Curve Plot The Melt Curve Plot displays the melt curve of the amplification products in the
overview selected wells.
Review the Melt Curve Plot to confirm that the amplification products in a well
display a single melting temperature (Tm). Multiple peaks in a melt curve indicate
non-specific amplification or primer-dimer formation.

Table 27 Melt Curve plots

Plot Description

Derivative Reporter vs. Displays the derivative reporter signal in the y-axis as a
Temperature function of temperature.
The peaks in the plot indicate significant decrease in
SYBR™ Green signal, and therefore the Tm of the amplified
products. Use this plot to confirm a single Tm of the
amplification products.

Normalized Reporter vs. Displays the fluorescence signal from the reporter dye
Temperature normalized to the fluorescence signal of the passive
reference, as a function of temperature.
You can use this plot to check the quality of the
fluorescence data.

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8 Chapter 8 Set up, run, and review melt curve experiments
Review results

View and assess You can view and assess the Melt Curve Plot in the Results tab.
the Melt Curve If no data are displayed in the Results tab, click Analyze.
Plot
1. In the Results tab, select Melt Curve Plot from the dropdown list.

2. Click to configure the plot, making the following selections:

• Plot Type: Derivative Reporter
• Color: Sample, Target, or Well
• Target: All or a target of interest
• (For custom experiments with more than one Melt Curve stage) Select the Melt
Curve stage to view.
The Melt Curve Plot is displayed for the selected wells of the selected stage.

3. Review the plot for evidence of unexpected multiple peaks, which can indicate
non-specific amplification or formation of primer-dimers.

4. Review the Well Table for the calculated Tm in each well.

Figure 16 Example Melt Curve Plot

Melt curve Use the Melt Curve Settings tab to enable or disable the Multi-Peak Calling function
settings overview and adjust the detection levels for additional peaks, if needed.
• Enable or disable Multi-Peak Calling.

Multi-Peak Calling Description

Enabled – More than one PCR product is expected to amplify.
– Tm will be determined for more than one peak.

Disabled – A single PCR product is expected to amplify.

– Tm will be determined for one peak.

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 Chapter 8 Set up, run, and review melt curve experiments
Review results 8

• (For multi-peak calling only) Adjust the detection levels for additional peaks.

Option Description
Peak level relative to Specify a fractional-level value as the additional peak
the dominant peak (%) detection threshold. The detected peaks are measured
relative to the height of the tallest peak, which has a
perfect fractional level of 100%. The default value is 10%.
For example, set a fractional-level detection threshold
value at 40, then peaks above 40% of the tallest peak are
reported, and peaks below 40% are regarded as noise.
Peak Calling Threshold Specify an absolute fluorescence-level value as the peak
calling threshold. The absolute fluorescence is measured
on the derivative reporter (-dRn') axis. Only peaks that
appear above the peak calling threshold will be detected.
For example, set a fluorescence-level value at 90,000,
then peaks with fluorescence above 90,000 are reported,
and peaks below 90,000 are regarded as noise.

For step-by-step instructions for adjusting the melt curve settings, see the desktop
software Help.

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 A Instrument overview

■ Power on the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

■ View run history and delete or transfer files from the instrument . . . . . . . . . . . 80
■ Load and unload the plate in the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Power on the instrument

1. Touch anywhere on the touchscreen to determine if the instrument is in sleep
mode. If the home screen is displayed, the instrument is already powered on.

2. If the home screen does not display,

power on the instrument by pressing the
switch on the rear panel.

If left unattended (for about two hours), the

instrument automatically enters sleep mode
(enabled by default) to conserve power.
Note: To customize the sleep mode setting,
touch Settings4Instrument
Settings4Sleep Mode.

View run history and delete or transfer files from the instrument
In the home screen, touch Settings4Run History.
• Touch an individual run record to view its details, then complete one of the
following actions:
– Touch Delete to delete the run record.
– Touch Transfer to export the run data.
• Touch Manage to select multiple run records for simultaneous viewing, deletion,
or transfer.
Note:
· Guests (users not signed-in) can only view guest run records.
· Users signed into their instrument profiles can also view their own run records.
· Administrators can view all run records.
Note: If the connection between the instrument and the desktop software is
interrupted during the run, the instrument still completes the run. However, the run
data (EDS file) must be transferred from the instrument to the desktop software using
a USB drive or a network drive.

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 Appendix A Instrument overview
Load and unload the plate in the instrument A

Load and unload the plate in the instrument

Load and unload a CAUTION! Use flat caps for 0.2-mL tubes. Rounded caps can damage the
plate in the heated cover.
QuantStudio™ 1
Real-Time PCR 1. Load the plate.
Instrument a. Open the instrument drawer.

b. Load the plate onto the plate adapter so that the following criteria are met.
• Well A1 of the plate is in the top-left corner of the plate adapter.
• The barcode faces the front of the instrument.

Note: Do not remove the black plate adapter before loading a plate or tube
strips. If used, tube strips can fit loosely in the adapter, but the heated cover
will apply the appropriate pressure to seat the tube strips securely in the
adapter.

c. Close the instrument drawer.

2. When the run ends, unload the plate.

a. Open the instrument drawer.

b. Remove the plate.

c. Close the instrument drawer.

CAUTION! PHYSICAL INJURY HAZARD. During instrument operation,

the plate temperature can reach 100°C. Allow it to cool to room
temperature before handling.

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A Appendix A Instrument overview
Load and unload the plate in the instrument

Load and unload a CAUTION! Use flat caps for 0.2-mL tubes and 0.1-mL tubes. Rounded caps can
plate in the damage the heated cover.
QuantStudio™ 3
Real-Time PCR 1. Load the plate.
Instrument or a. Touch to eject the instrument drawer.
QuantStudio™ 5
Real-Time PCR b. Load the plate onto the plate adaptor so that the following criteria are met.
Instrument • Position well A1 of the plate in the top-left corner of the plate adapter.
• Ensure that the barcode faces the front of the instrument.

IMPORTANT! The instrument should be used by trained operators who

have been warned of the moving parts hazard.

Note: (For 96-well 0.2-mL blocks only) Do not remove the black plate adapter
before loading a plate or tube strips. If used, tube strips can fit loosely in the
adapter, but the heated cover will apply the appropriate pressure to seat the
tube strips securely in the adapter.
Note: The 384-well and 96-well Fast (0.1-mL) block configurations do not
require a plate adapter.

c. Touch to close the instrument drawer.

2. When the run ends, unload the plate.

a. Touch to eject the instrument drawer.

b. Remove the plate.

c. Touch to close the instrument drawer.

CAUTION! PHYSICAL INJURY HAZARD. During instrument operation,

the plate temperature can reach 100°C. Allow it to cool to room
temperature before handling.

Note: If the instrument does not eject the plate, contact Support.

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 B Alternative procedures to set up a
template

■ Set up a custom experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

■ Assign samples using a sample definition file . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
■ Assign samples and targets using plate setup files . . . . . . . . . . . . . . . . . . . . . . . . 86
■ Assign targets, samples, and biological replicate groups from an XLS file . . . . 87
■ Create new EDT files using existing EDT and EDS files . . . . . . . . . . . . . . . . . . . . 87

Set up a custom experiment

Custom Custom experiment setup is required for assays that use multiple PCR stages, such as
experiments TaqMan® Mutation Detection Assays. A custom experiment also allows flexibility for
secondary analysis.
overview
The default settings for custom experiments are that of a standard curve experiment,
but most settings are editable.

Table 28 Default settings in custom experiments

Setting Default

Run method (thermal protocol) Equivalent to standard curve experiment default

Tasks Unknown
Negative control
Standard

Ct settings Baseline threshold equivalent to standard curve

experiment default

Flag settings QC flags on

No automatic omissions

Auto Export Off

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B Appendix B Alternative procedures to set up a template
Set up a custom experiment

Table 29 Editing options in custom experiments

Setting Description

Run method (thermal protocol) • Ramp rates can be edited within software limits.
• Data collection can be enabled at any step and
during any ramp within a melt stage.
• Multiple instances of any type of stage can be
added, with exceptions noted.
• Any stage can be added at any point in a run
method, with exceptions noted.
Noted exceptions:
• Only one infinite hold (must be added at the end).
• Only one pre‑PCR read and one post‑PCR read
stage. If both exist in a run method, the pre‑read
must be before the post‑read.
For example, the following order is valid:
melt–PCR–Pre‑Read–Melt–PCR.

Analysis settings Editable

Flags Editable

Optical filters Editable

Set up a custom 1. In the Home screen, create or open a template.

experiment in the • In the New Experiment pane, click Create New Experiment to create a
software new template.
• In the Open Existing Experiment pane, click Open to select and open an
existing template.

2. In the Properties tab, enter the template information.

3. In the Method tab, edit the default run method according to the experiment
requirements.

4. In the Plate tab (Quick Setup), assign plate attributes.

a. In the Plate Attributes pane, select the Passive Reference from the
dropdown list.

5. In the Plate tab (Quick Setup), define and assign well attributes.
a. Select wells in the Plate Layout or the Well Table.

b. Assign samples and targets to selected wells.

• Enter new sample and target names in the text fields.
• Select previously defined samples and targets from the dropdown lists.
Note: New sample or target names entered in the Quick Setup subtab are
automatically populated with default values for Reporter (FAM) and
Quencher (NFQ-MGB) and assigned a task ( Unknown). Edit these
values in the Advanced Setup subtab.

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 Appendix B Alternative procedures to set up a template
Assign samples using a sample definition file B

6. (Optional) In the Plate tab (Advanced Setup), assign tasks.

a. Select wells in the Plate Layout or the Well Table.

b. In the Targets table, select the checkbox of a target, then select a task from
the dropdown list.

7. (Optional) In the Plate tab (Advanced Setup), define and assign biological
replicate groups (see “Define and assign biological replicate groups“ on
page 95).

Assign samples using a sample definition file

About sample Import sample information from a sample definition file to include in the plate setup.
definition files A sample definition file is a CSV file or a TXT file that contains the following setup
information:
• Sample name
• (Optional) Custom sample properties

Create a sample 1. In a spreadsheet program, create the following column headers:

definition file • Well
• Sample Name
• (Optional) Column header names for up to 32 user-defined custom fields (for
example, Custom 1, Custom 2, etc.)

2. Enter the well number and sample name in the appropriate columns.

3. (Optional) Enter the custom properties for the sample.

4. Save the file as a tab-delimited text file (TXT) or a comma-separated values file
(CSV).

Import sample Example setup files are provided with the software in:
information from a <drive> :\Program Files (x86)\Applied Biosystems\QuantStudio
sample definition Design and Analysis Software\examples\User Sample Files,
file where <drive> is the drive on which the software is installed.

1. In an open experiment, select File4Import Plate Setup.

2. Click Browse, navigate to a sample definition text file, then click Select.

3. Click Apply.

4. If the experiment already contains plate setup information, the software prompts
for the replacement of the plate setup with the data from the file. Click Yes to
replace the plate setup information.

The samples appear in the Samples table for the experiment. All samples and well
assignments in the experiment are replaced with those in the file. If defined, the
custom sample properties also appear in the Well Table of the Results tab and in

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B Appendix B Alternative procedures to set up a template
Assign samples and targets using plate setup files

the Plate Layout tooltips in both the Plate and Results tabs. The custom fields can
be exported with the results data.
Note: To modify custom sample properties information, edit the custom fields in the
sample definition file and import the file again. All of the sample information in the
experiment is replaced with the information in the new file.

Assign samples and targets using plate setup files

About plate setup Plate setup files contain setup information such as the well number, sample name,
files sample color, target name, dyes, and other reaction plate contents.
Plate setup files can be exported from previously run experiments. For instructions on
exporting an experiment, see “Export experiments or results“ on page 40.

Import plate setup Import the plate setup for a new experiment from an exported file with one of the
data following formats:
• EDS – EDS file format
• EDT – user-created and system templates files format
• TXT – text format
• XML – XML format
• CSV – comma separated values format
• SDT – Sequence Detection System (SDS) template files format
• SDS – 7900 v2.4 format
Note: Import plate setup information from a 96-well plate into a 384-well plate,
provided that the sample file is a TXT file.

IMPORTANT! The file must contain only plate setup data and it must match the
experiment type.

1. In the Plate tab, select File4Import Plate Setup.

2. Click Browse, navigate to and select the file to import, then click Select.
Example setup files are provided with the software in:
<drive> :\Program Files (x86)\Applied Biosystems\QuantStudio
Design and Analysis Software\examples\User Sample Files,
where <drive> is the drive on which the software is installed.

3. Click Apply.
The setup data from the selected file is imported into the open experiment.

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 Appendix B Alternative procedures to set up a template
Assign targets, samples, and biological replicate groups from an XLS file B

Assign targets, samples, and biological replicate groups from an

XLS file
For wells with single targets, you can paste assignment information from an XLS file
into the plate layout of the desktop software.
An example copy and paste file is provided with the software in:
<drive> :\Program Files (x86)\Applied Biosystems\QuantStudio
Design and Analysis Software\examples\User Sample Files.
where <drive> is the drive on which the software is installed.

1. In the custom properties tab of the example Microsoft™ Excel™ file, ensure that
the Well column is sorted in order 1 through 96, then select the Well column and
the Sample Name column, including headers.

2. In the Plate tab of the software, click Well Table, then ensure that the well
numbers are in order from 1 through 96.

3. In the Well Table, hover the mouse in the first cell underneath the Sample
header (adjacent to A1), right-click, then select either Paste or Paste only
samples.
Any of the columns not copied are treated as NULL values for those columns.

Create new EDT files using existing EDT and EDS files
About experiment Use templates to create experiments with the same parameters or with pre-existing
templates settings. Experiments can be saved as unlocked or locked (password-protected)
templates.
You can save the following information in an experiment template (EDT) file:
• Plate setup information (defined sample and targets or SNP assays, plate
assignment of samples and targets or SNP assays)
• Reagent information
• Run method (thermal protocol)
• Analysis settings
Example templates are provided with the software in:
<drive>:\Program Files (x86)\Applied Biosystems\QuantStudio
Design & Analysis Software\templates,
where <drive> is the drive on which the software is installed.

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 C Detailed procedures
to create or edit a method

■ Adjust method parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

■ Set up advanced temperature zones (Auto Delta and VeriFlex™ Zones) . . . . . . 89
■ Add or adjust a pause step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
■ Select optical filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
■ Edit the ramp increment for the melt curve dissociation step . . . . . . . . . . . . . . . 91

Adjust method parameters

For an overview of the method as it is graphically represented, see “Method
elements“ on page 18.

• In the Method tab, click a method parameter field to edit the following
information:
• Reaction volume
• Temperature ramp rate
• Step temperature
• Step hold time
• Number of cycles

• Click-drag to increase or decrease a step temperature.

• Click to switch data collection on or off at each step.

Data Collection On enables analysis of data that is collected throughout the PCR,
for real-time analysis and troubleshooting.

• (QuantStudio™ 3 Real-Time PCR System and QuantStudio™ 5 Real-Time PCR System

only) Click to configure settings for Auto Delta or VeriFlex™ Zones for
individual steps (see “Set up advanced temperature zones (Auto Delta and
VeriFlex™ Zones)“ on page 89).
Note: In melt curve stages, Advanced Settings are not applicable.
An or is displayed in the PCR stage when Auto Delta or VeriFlex™,
respectively, is enabled.

• Click to configure pause settings.

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 Appendix C Detailed procedures to create or edit a method
Set up advanced temperature zones (Auto Delta and VeriFlex™ Zones) C

• Adjust the heated cover temperature via the instrument settings (see
QuantStudio™ 1 Real-Time PCR System Installation, Use, and Maintenance Guide
(Pub. No. MAN0017853) or QuantStudio™ 3 and 5 Real-Time PCR Systems
Installation, Use, and Maintenance Guide (Pub. No. MAN0010407)).

• See the Help to learn more about adjusting the following parameters:
• Adding or subtracting a stage
• Adding or subtracting a step from a stage
• Configuring optical filter settings

Set up advanced temperature zones (Auto Delta and VeriFlex™

Zones)
(QuantStudio™ 3 Real-Time PCR System and QuantStudio™ 5 Real-Time PCR System only)
Configure settings for Auto Delta and VeriFlex™ Zones.
• Auto Delta—Incremental increase or decrease of a cycle's temperature or hold
time for a step in a cycling stage (not applicable for Hold or Infinite Hold stages).
• VeriFlex™ Zones—Independent temperature zones within 5°C of adjacent zones.
– QuantStudio™ 3 Real-Time PCR Instrument: 3 zones
– QuantStudio™ 5 Real-Time PCR Instrument: 6 zones
Note: VeriFlex™ Zones temperature settings are not available for 384-well blocks.

1. In the Method tab, click Advanced Settings in a step.

Note: Any changes apply only to the step in which you clicked.

2. Configure either the VeriFlex™ Zones or Auto Delta for the selected step.
• Select VeriFlex™, then enter a temperature for each zone.
Note: In the Plate tab, the VeriFlex™ Zones display on the plate layout.
· To view setting details, hover over the V in each zone.
· To hide the display of zones, click Action4Hide VeriFlex™ Zones.
• Select Auto Delta, then enter a starting cycle, temperature, and time.

3. Click Save.

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C Appendix C Detailed procedures to create or edit a method
Add or adjust a pause step

Add or adjust a pause step

1. In the Method tab, click in the step.

2. Select Pause.

3. Enter the cycle after which the pause should occur.

4. Enter a pause temperature between 4°C and 99.9°C.

CAUTION! PHYSICAL INJURY HAZARD. During instrument operation,

the plate temperature can reach 100°C. If you want to access the plate
during a run pause, enter room temperature as the pause temperature and
allow the plate to cool to room temperature before handling.

5. Click outside of the pause dialog box to return to the method.

6. (Optional) To remove a pause, click , then deselect Pause.

Select optical filters

The need to edit optical filter settings is rare, and it is intended for advanced or
custom uses only.
Use the optical filters settings feature to select a filter set to match the profile of a
custom dye.

1. In the Method tab, select Action4Optical filter settings.

The excitation (x) and emission (m) wavelengths that correspond to each filter are
shown on the screen.

2. Select the check boxes to enable or disable filters.

A Melt Curve Filter table is accessible if the method contains a melt curve stage.
Otherwise, use the PCR Filter table to select optical filters.

3. (Optional) Click Revert to Defaults to reset filters.

4. Click Close.

For information on the dyes read by each filter, see QuantStudio™ 1 Real-Time PCR
System Installation, Use, and Maintenance Guide (Pub. No. MAN0017853) or
QuantStudio™ 3 and 5 Real-Time PCR Systems Installation, Use, and Maintenance Guide
(Pub. No. MAN0010407)

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 Appendix C Detailed procedures to create or edit a method
Edit the ramp increment for the melt curve dissociation step C

Edit the ramp increment for the melt curve dissociation step
In the Method tab, you can perform the following tasks to edit the ramp increment for
the melt cure dissociation step.

• Select the ramp increment method for the dissociation step (located under the
graphical representation of the thermal protocol).
Option Description
Continuous (default) Continuously increases the temperature by the ramp
increment (°C/sec).
Step and Hold Increases the temperature by the ramp increment (°C),
then holds at that temperature for the specified time.
No. of Data Points per Increases the temperature by the ramp increment (°C)
Degree and collects the specified number of data points per
degree increased.

• (For all options) Edit the temperature ramp increment.

a. Click the ramp increment element in the Dissociation step.

b. Enter a value or use the up/down arrows (default is 0.15°C/s).

• (Step and Hold only) Edit the hold time after each temperature increase.
a. Click the time field next to Step and Hold.

b. Enter a value or use the up/down arrows (default is 5 seconds).

• (No. of Data Points per Degree only) Edit the number of data points to be collected
with each degree increase.
a. Click the number of data points element in the Dissociation step.

b. Enter a value or use the up/down arrows (default is 10 data points).

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 D Detailed procedures to set up
plate / well details and libraries

■ Assign well attributes (Quick Setup subtab) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

■ Define and assign well attributes (Advanced Setup subtab) . . . . . . . . . . . . . . . . 93
■ Assign a task to wells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
■ Define and set up standard dilutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
■ Define and assign biological replicate groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
■ Sample, target, and SNP assay libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

Assign well attributes (Quick Setup subtab)

In the Quick Setup subtab, assign well attributes by direct entry into text fields or by
selecting user-defined samples and targets or SNP assays from dropdown lists.

1. In the Plate tab, click Quick Setup.

2. Select plate wells in the Plate Layout or the Well Table.

3. Assign the well attributes for the selected wells.

• Into the text fields, enter the names of the new sample and the new target or
SNP assay.
• From the dropdown lists, select a user-defined sample and target or SNP
assay.
For more information about defining or importing samples and targets or
SNP assays, see “Define and assign well attributes (Advanced Setup subtab)“.
Note: In the Advanced Setup subtab, change the default selections for the
reporter and quencher dyes and for tasks (see “Assign a task to wells“ on
page 93).

4. (Optional) Enter comments for the selected wells.

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 Appendix D Detailed procedures to set up plate / well details and libraries
Define and assign well attributes (Advanced Setup subtab) D

Define and assign well attributes (Advanced Setup subtab)

In the Advanced Setup subtab, define or import samples and targets or SNP assays,
then assign well attributes.

1. In the Plate tab, click Advanced Setup.

2. In the Samples table, define samples (see “Define samples in the Samples
table“ on page 97).

3. In the Targets or SNP Assays table, define targets or SNP assays, then select
detection tasks.
a. Define targets or SNP assays (see “Define targets in the Targets table“ on
page 98 or “Define SNP assays in the SNP Assays table“ on page 99,
respectively).

b. Select a detection task from the Task column dropdown list (see “Assign a
task to wells“ on page 93).

4. Assign well attributes.

a. Select plate wells in the Plate Layout or the Well Table (see “Select
plate wells“ on page 19).

b. Select the checkbox of a defined sample.

c. Select the checkbox of a defined target or SNP assay.

Assign a task to wells

1. In the Plate tab, click Advanced Setup.

2. Select plate wells in the Plate Layout or the Well Table (see “Select plate
wells“ on page 19).

3. In the Targets or SNPs table, select the check box of a target or SNP assay.

4. Select a detection task from the Task column dropdown list.

Detection tasks for targets and SNP assays

Task Description

Unknown (default) The well contains test samples with unknown genotype.

Negative Control / The well contains water or buffer instead of sample.

No template control

Standard [1] The well contains samples with known standard quantities.
Note: For a standard detection task, enter the standard quantity in the quantity column.

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 D Appendix D Detailed procedures to set up plate / well details and libraries
Define and set up standard dilutions

Task Description

Positive Control The well contains samples homozygous for allele 1.

Allele 1 / Allele 1 [2]

Positive Control The well contains samples homozygous for allele 2.

Allele 2 / Allele 2 [2]

Positive Control The well contains samples heterozygous for allele 1 and 2.
Allele 1 / Allele 2 [2]

Internal positive The PCR reaction contains a short synthetic DNA template to distinguish between true
control [3] negative results (that is, the target is absent in the samples) and negative results caused
by PCR inhibitors, incorrect assay setup, or reagent or instrument failure.

Blocked IPC [3] The well contains an IPC blocking agent, which blocks amplification of the IPC.

NAC – No amplification The PCR reaction contains an IPC blocking agent instead of sample.
control [3]
No amplification should occur in negative control-blocked IPC wells because the reaction
contains no sample and amplification of the IPC is blocked.
[1] For standard curve and relative standard curve experiments only.
[2] For genotyping experiments only.
[3] For presence/absence experiments only.

Define and set up standard dilutions

Note: This information is applicable for standard curve and relative standard curve
experiments only.

1. In the Plate tab, select Action4Define and Set Up Standards.

2. Select Singleplex or Multiplex from the Model dropdown list.

3. (Optional) Select the target from the dropdown list.

4. Enter the parameters for the dilution series.

• Number of dilution points—5 recommended
• Number of replicates—3 recommended
• Starting Quantity—The highest or lowest standard quantity, without units.
Note: The quantity can be expressed as copies, copies/µL, ng/µL, or as
relative dilutions.
Note: Use E to indicate the exponent number using scientific notation. For
example, to indicate 1.23 × 104, enter 1.23E4.
• Serial Factor
Note: The serial factor calculates quantities for all standard curve points.
– Starting quantity is the highest value—Select 1:10 to 1:2.
– Starting quantity is the lowest value—Select 2× to 10×.
Note: The Standard Curve Preview y-axis values are calculated from the
starting quantity and serial factor. Actual results may differ from the preview.

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 Appendix D Detailed procedures to set up plate / well details and libraries
Define and assign biological replicate groups D

5. Select and arrange the wells to use for the standards.

• Select Automatically Select Wells for Me.
• Select Let Me Select Wells, then select wells using the displayed plate layout.

6. Select to arrange the standards in Columns or Rows.

7. (Optional) Click Reset to revert to default values.

8. Click Apply, then click Close to return to the Plate tab.

Assign the Note: Applicable for standard curve and relative standard curve experiments only.
standard dilutions 1. In the Plate tab, select wells in the Plate Layout or Well Table.
manually
2. Select the check box for the target, select from the Task dropdown list, then
enter a quantity.

3. Repeat to complete the standard dilution series.

Define and assign biological replicate groups

Biological Replicate Groups can be used in standard curve, relative standard curve,
comparative Ct, and custom experiments.

1. In the Plate tab, click Advanced Setup.

2. Define Biological Replicate Groups.

a. In the Biological Replicate Groups table, click Add.

b. (Optional) Click a cell to edit color, name, or comments.

c. (Optional) Click to delete a biological replicate group from the table.

3. Assign Biological Replicate Groups.

a. Select plate wells in the Plate Layout or the Well Table (see “Select
plate wells“ on page 19).

b. In the Biological Replicate Groups table, select the check box of a biological
replicate group.

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D Appendix D Detailed procedures to set up plate / well details and libraries
Sample, target, and SNP assay libraries

Sample, target, and SNP assay libraries

Libraries overview Libraries contain saved information to reuse in future templates.
The following libraries are available in the software:
• Dye Library
• Sample Library
• Target Library
• SNP Assay Library
• Analysis Settings Library
To access the libraries, use the Tools menu or the Plate tab.
• In the menu bar, select Tools4(Library of choice).
• In the Plate tab of an open EDT or EDS file, click Advanced Setup, then select
Action4Import from Library.

Apply a filter to You can filter the Sample, SNP assay, Target, and Analysis Settings Libraries.
search a library 1. Access a library of interest.
• In the menu bar, select Tools4(Library of interest).
• In the Plate tab of an open EDT or EDS file, click Advanced Setup, then select
Action4Import from Library.

2. Select a feature from the first dropdown list. Each column of the table is an
available feature.

3. Select a condition from the second dropdown list to define the feature. The
conditions will vary by feature.

4. Enter a value or text by which to filter.

5. Click Apply Filter.

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 Appendix D Detailed procedures to set up plate / well details and libraries
Sample, target, and SNP assay libraries D

Define samples in In the Plate tab, click Advanced Setup, then perform one of the following actions in
the Samples table the Samples table.
Option Action
Manually define a 1. Click Add.
sample
2. Click a cell to edit the attributes for the sample.
3. (Optional) Click in the table header to add a Custom
Attribute column.
a. Click the Custom Attribute column header, then edit the
header with a new sample attribute.
b. Click a Custom Attribute cell in the table, then enter the
attribute information.

Import samples 1. Select Action4Import from File.

from a
TXT or XLS file 2. Navigate to and select a file, then click Open.

Import samples 1. Select Action4Import from Library.

from the Sample
Library 2. (Optional) Apply a filter to search for a specific sample (see
page 96).
3. Select one or more samples, then click Add Selected.
Note: Shift‑click or Ctrl‑click to select multiple samples.

Save a sample to the Select a sample row, then select Action4Save to Library.
Sample Library
Delete a sample Select a sample row, then click .
from the table

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D Appendix D Detailed procedures to set up plate / well details and libraries
Sample, target, and SNP assay libraries

Define targets in In the Plate tab, click Advanced Setup, then perform one of the following actions in
the Targets table the Targets table.
Option Action
Manually define a 1. Click Add.
target
2. Click a cell to edit the attributes for the target.

Import targets from a 1. Select Action4Import from Library.

TXT or XML file
2. Click Import or Import AIF.
3. Navigate to and select a file, then click Import.
4. Select one or more targets, then click Add Selected.
Note: Shift‑click or Ctrl‑click to select multiple targets.

Import targets from 1. Select Action4Import from Library.

the Target Library
2. (Optional) Apply a filter to search for a specific target (see
page 96).
3. Select one or more targets, then click Add Selected.
Note: Shift‑click or Ctrl‑click to select multiple targets.

Save a target to the Select a target row, then select Action4Save to Library.
Target Library
Delete a target from Select a target row, then click .
the table

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 Appendix D Detailed procedures to set up plate / well details and libraries
Sample, target, and SNP assay libraries D

Define SNP assays In the Plate tab, click Advanced Setup, then perform one of the following actions in
in the SNP Assays the SNP Assays table.
table Option Action
Manually define a SNP 1. Click Add.
assay
2. Click a cell to edit the attributes for the SNP assay.

Import SNP assays from 1. Select Action4Import from Library.

a TXT or XML file
2. Click Import or Import AIF.
3. Navigate to and select a file, then click Import.
4. Select one or more SNP assays, then click Add Selected.
Note: Shift‑click or Ctrl‑click to select multiple SNP
assays.

Import SNP assays from 1. Select Action4Import from Library.

the SNP Assay Library
2. (Optional) Apply a filter to search for a specific SNP assay
(see page 96).
3. Select one or more SNP assays, then click Add Selected.
Note: Shift‑click or Ctrl‑click to select multiple SNP
assays.

Save a SNP assay to the Select a SNP assay row, then Action4Save to Library.
SNP Assay Library
Delete a SNP assay Select a SNP assay row, then click .
from the table

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 E Configure analysis settings

■ Guidelines for the analysis settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

■ View and configure the analysis settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
■ Ct settings overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
■ Flag settings overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
■ Advanced settings overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

This section describes the analysis settings that apply to all experiment types, unless
otherwise noted.

Guidelines for the analysis settings

• We recommend analyzing the experiment with the default analysis settings.
• If the default analysis settings are not suitable for the experiment, modify the
settings in the Analysis Settings dialog box, then reanalyze the experiment.
• Save modified analysis settings to the Analysis Settings Library.
The default analysis settings are different for each experiment type. The analysis
settings determine for following parameters.
• How the baseline, threshold, and threshold cycle (Ct) are calculated
• Which flags are enabled
• Other analysis options that are specific to an experiment type
For detailed information about different types of analysis settings, see the following
sections.
• “Ct settings overview“ on page 101
• “Flag settings overview“ on page 102
• “Advanced settings overview“ on page 102
• “Standard curve settings overview“ on page 47
• “Relative quantification settings overview“ on page 59
• “Call settings overview (genotyping)“ on page 66
• “Call settings overview (presence / absence)“ on page 73
• “Melt curve settings overview“ on page 78

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 Appendix E Configure analysis settings
View and configure the analysis settings E

View and configure the analysis settings

1. In the Results tab, click (in top-right corner).

2. View and (optional) configure the analysis settings.

For step-by-step instructions for adjusting analysis settings, see the desktop
software Help.

3. Click Apply.

4. Click Analyze to reanalyze to experiment with the new settings.

5. (Optional) To save the settings in the Analysis Settings Library, click Save.

6. (Optional) To return to the default settings, click Revert.

Ct settings overview
The default Ct settings are appropriate for most applications. Configuration of the
settings is an option for analysis of atypical or unexpected run data.
For step-by-step instructions for adjusting the Ct settings, see the desktop software
Help.
Note: The Ct Settings feature is not available for experiments without a PCR stage,
such as melt curve experiments.

Table 30 Ct Settings

Setting Description

Data Step Selection Determines the stage/step combination for Ct analysis (when there is more than one data
collection point in the run method).

Algorithm Settings – The Baseline Threshold Algorithm is used to calculate the Ct values.
Baseline Threshold
This algorithm is an expression estimation algorithm that subtracts a baseline
component and sets a fluorescence threshold in the exponential region.

Algorithm Settings – The Relative Threshold (Crt) Algorithm is used to calculate the Crt values.
Relative Threshold

Default Ct Settings Determines how the Baseline Threshold Algorithm is set. The Default Ct Settings are
used for targets unless they have custom settings.
For recommendations on adjusting baseline and threshold settings, see Table 31.

Ct Settings for Target • Default Settings selected—The Default Ct Settings are used to calculate the Ct
values for the target.
• Default Settings deselected—The software allows manual setting of the baseline or
the threshold.
For recommendations for adjusting baseline and threshold settings, see Table 31.

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E Appendix E Configure analysis settings
Flag settings overview

Table 31 Recommendations for manual threshold and baseline settings

Setting Recommendation

Threshold Enter a value for the threshold so that the threshold is:
• Above the background.
• Below the plateau and linear phases of the amplification curve.
• Within the exponential phase of the amplification curve.

Baseline Select the Start Cycle and End Cycle values so that the baseline ends
before significant fluorescence signal is detected.

Flag settings overview

Use the Flag Settings to configure the following parameters.
• Adjust the sensitivity so that more wells or fewer wells are flagged.
• Change the flags that are applied by the software for each experiment type.
For step-by-step instructions for configuring the flag settings, see the desktop software
Help.

Advanced settings overview

Use the Advanced Settings tab to change baseline settings for individual wells.
For step-by-step instructions for adjusting the advanced settings, see the desktop
software Help.
Note: The Advanced Settings feature is not available for experiments without a PCR
stage, such as melt curve experiments.

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 F Documentation and support

■ Related documentation for the QuantStudio™ 1 Real-Time PCR System . . . . 103

■ Related documentation for the QuantStudio™ 3 Real-Time PCR System
and QuantStudio™ 5 Real-Time PCR System . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
■ Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
■ Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

Related documentation for the QuantStudio™ 1 Real-Time PCR

System
Document Publication number

QuantStudio™ 1 Real-Time PCR System Installation, Use, and MAN0017853

Maintenance Guide

QuantStudio™ Design and Analysis Desktop Software MAN0010409

Command-Line Application Guide

QuantStudio™ Design and Analysis Desktop Software User MAN0010408

Guide

QuantStudio™ 1 Real-Time PCR System Site Preparation MAN0017854

Guide

QuantStudio™ Design and Analysis Desktop Software User Guide 103

F Appendix F Documentation and support
Related documentation for the QuantStudio™ 3 Real-Time PCR System and QuantStudio™ 5 Real-Time PCR System

Related documentation for the QuantStudio™ 3 Real-Time PCR

System and QuantStudio™ 5 Real-Time PCR System
Document Publication number

QuantStudio™ 3 and 5 Real-Time PCR Systems Installation, MAN0010407

Use, and Maintenance Guide

QuantStudio™ Design and Analysis Desktop Software MAN0010409

Command-Line Application Guide

QuantStudio™ Design and Analysis Desktop Software User MAN0010408

Guide

QuantStudio™ 5 Real-Time PCR System SAE Admin Console MAN0010410

User Guide

QuantStudio™ 3 and 5 Real-Time PCR Systems Site MAN0010405

Preparation Guide

Customer and technical support

Visit thermofisher.com/support for the latest service and support information.
• Worldwide contact telephone numbers
• Product support information
– Product FAQs
– Software, patches, and updates
– Training for many applications and instruments
• Order and web support
• Product documentation
– User guides, manuals, and protocols
– Certificates of Analysis
– Safety Data Sheets (SDSs; also known as MSDSs)
Note: For SDSs for reagents and chemicals from other manufacturers,
contact the manufacturer.

Limited product warranty

Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth
in the Life Technologies' General Terms and Conditions of Sale at
www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you have
any questions, please contact Life Technologies at www.thermofisher.com/support.

104 QuantStudio™ Design and Analysis Desktop Software User Guide

 Glossary

biological Reactions that contain identical components and volumes, but evaluate separate
replicates samples of the same biological source (for example, samples from three different mice
of the same strain, or separate extractions of the same cell line or tissue sample).
For runs that use biological replicate groups in a gene expression project, the values
displayed in the biological replicates lists are calculated by combining the results of
the separate biological samples and treating this collection as a single population (that
is, as one sample).
For ΔCt computations (normalizing by the endogenous control) in a singleplex
experiment, the software averages technical replicates. The averages from the
technical replicates are then averaged together to determine the value for that
biological replicate.

endogenous A gene that is used to normalize template differences and sample-to-sample or run-to-
control run variation.

endpoint read See post-PCR read.

post-PCR read In genotyping and presence/absence experiments, the part of the instrument run that
occurs after amplification. In genotyping experiments, fluorescence data collected
during the post-PCR read are displayed in the allelic discrimination plot and used to
make allele calls. In presence/absence experiments, fluorescence data collected during
the post-PCR read are displayed in the presence/absence plot and used to make
detection calls. Also called endpoint read.

pre-PCR read In genotyping and presence/absence experiments, the part of the instrument run that
occurs before amplification. The pre-PCR read is optional but recommended.
Fluorescence data collected during the pre-PCR read can be used to normalize
fluorescence data collected during the post-PCR read.

reference sample In relative standard curve and comparative Ct (ΔΔCt) experiments, the sample used as
the basis for relative quantification results. Also called the calibrator.

target The nucleic acid sequence that is amplified and detected during PCR.

task In the software, the type of reaction performed in the well for the target.

technical Reactions that contain identical components and volumes, and that evaluate the same
replicates sample; important for evaluating precision.

QuantStudio™ Design and Analysis Desktop Software User Guide 105

 Index

A Baseline Threshold Algorithm 101

biological replicate groups, define and assign 95
absolute quantity 41
accessories 21
Advanced Setup 93 C
advanced temperature zones 89 calibration data, override 39
algorithm settings calibration results, view 38
Baseline Threshold Algorithm 101 column in XLS file, well number 87
Relative Threshold Algorithm 101 comparative Ct experiments
Allelic Discrimination Plot, overview 64 compared to relative standard curve 51
Amplification Plot endogenous control 50
baseline 30 Gene Expression Plot 57
baseline setting 32 negative controls 50
exponential phase 30 overview and purpose 50
linear phase 30 PCR options 50
optimize to view negative controls 34 QC Plot 58
overview 30 reaction types 50
plateau phase 30 reference genes 50
shape 30 relative quantification settings 59
threshold settings 31 replicates 50
analysis settings review results workflow 54
advanced settings 101, 102 set up in software 53
configure 100 consumables 21
flag settings 101, 102 Cq vs Ct vs Crt 26
guidelines 100 Ct precision, improve 33
melt curve settings 78 Ct settings
standard curve settings 101 algorithm settings 101
Analysis Settings Library 96
default 101
assign select data step 101
biological replicate group 95 tab 101
sample 18, 92, 93 target 101
SNP assay 18, 92, 93 custom dye 17
target 18, 92, 93 custom experiments
task 18, 92, 93 overview 83
well details 92 set up in software 84
assign standard dilutions 95
assign targets, samples, and biological replicates,
from XLS file 87 D
Auto Delta, settings 89 define
auto-analysis 29 biological replicate group 95
automatic threshold 101 samples 97
SNP assays 99
B targets 98
delete files 80
barcode scan 17 documentation, related 103, 104

106 QuantStudio™ Design and Analysis Desktop Software User Guide

 Index

Dye Library 96 L
dye signal accuracy, Multicomponent Plot 36
library
apply filter 96
E list of 92
limited product warranty 104
endogenous control
load plate 81, 82
comparative Ct experiments 58
locked template, save 20
relative standard curve experiments 58
example templates 87
experiment M
export 40
melt curve experiments
setup workflow 15
overview and purpose 75
experiment data, import 86
ramp increment 91
experiment template 87
reaction types 75
experiment types 11
reactions 77
experiments, define parameters 20
review results workflow 77
export
set up in software 76
experiment or results 40
settings 78
experiments or results 40
Melt Curve Plot
overview 77
F view and assess 78
method, create or edit 88
flags 38
monitor run 22
Multicomponent Plot
G dye signal accuracy 36
Gene Expression Plot view 36
comparative Ct experiments 57
overview 57 N
relative standard curve experiments 57
NULL values in well assignments 87
RQ vs Sample 57
RQ vs Target 57
genotyping experiments O
Allelic Discrimination Plot 64
omit wells 33
call settings 66
optical filter selection 17
manual call 65
optical filter settings 90
PCR options 61
Presence/Absence Plot 71
reaction types 61 P
review results workflow 63
parts and materials 21
set up in software 61
pause, add or adjust 90
PCR options
I comparative Ct experiments 50
genotyping experiments 61
identify well problems 35
presence/absence experiments 69
Index Term 13
relative standard curve experiments 49
instrument, power on 80
standard curve experiments 42
PCR reactions
K guidelines for set up 21
handle samples and reagents 21
kits 21
prepare 21
set up and run 44, 54, 63, 70
plate set up 18

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Index

plate setup data, import 86 PCR options 49

Plate tab 92 QC Plot 58
post-run summary 24 reaction types 49
preferences, set 14 relative quantification settings 59
presence/absence experiments replicates 49
call settings 73 review results workflow 54
IPC 68 set up in software 51
overview and purpose 68 standard dilution series 49
PCR options 69 Relative Threshold Algorithm 101
reaction types 68 required components
review results workflow 71 negative controls 41, 49, 61
set up in software 69 reference genes 49
Presence/Absence Plot replicates 41, 61
overview 71 sample types 41, 61
view and assess 72 standard 41
standard dilution series 41
results, export 40
Q Results tab 27
QC Plot review
comparative Ct experiments 58 Amplification Plot 30
overview 58 Multicomponent Plot 36
relative standard curve experiments 58 QC Summary 38
view and assess 58 Raw Data Plot 37
QC Summary Standard Curve Plot 45, 55
review flags 38 Well Table 35
view 38 run history 80
quantification cycle (Cq) 26 run results, review workflow 29
Quick Setup 18, 92

S
R sample
Raw Data Plot assign to well 18, 92, 93
determine signal accuracy 37 define 97
view 37 sample definition file
reaction types create 85
comparative Ct experiments 50 spreadsheet 85
genotyping experiments 61 sample information, import 85
melt curve experiments 75 Sample Library 96
presence/absence experiments 68 Samples table, manage 97
relative standard curve experiments 49 save template 20
standard curve experiments 41 select wells 19
reagents, enter information 17 signal accuracy, Raw Data Plot 37
real-time plots SNP assay
view on instrument touchscreen 23 assign to well 18, 92, 93
zoom 24 define 99
related documentation 103, 104 SNP Assay Library 96
relative quantification settings 59 SNP Assays table, manage 99
relative quantity 48, 50 standard 41, 49
relative standard curve experiments standard curve, use a different standard curve 47, 57
assign standard dilutions 95 standard curve experiments
compared to comparative Ct 51 assign standard dilutions 95
endogenous control 49 overview and purpose 41
Gene Expression Plot 57 PCR options 42
overview and purpose 48 reaction types 41

108 QuantStudio™ Design and Analysis Desktop Software User Guide

 Index

review results workflow 44 thermal protocol, create or edit 88

set up in software 43 transfer files 80
Standard Curve Plot
overview 45, 55
view and assess 46, 56 U
standard curve settings 47, 57 unload plate 81, 82
standard dilutions, set up 94 unlocked template, save 20
standards, define and set up 94
start run 22
support, customer and technical 104 V
VeriFlex, settings 89

T
target W
assign to well 18, 92, 93 warranty 104
define 98 well details
Target Library 96 assign 92
Targets table, manage 98 define 92
task view on instrument touchscreen 23
assign to well 18, 92, 93 Well Table
descriptions 93 group or sort 35
template overview 35
adjust reaction volume 17 wells, select 19
create 16 workflow
enter properties 16 review run results 29
information saved 87 run experiment 15
open 16 set up experiment 15
optical filter selection 17
set up 15
use default method 17 Z
terms and conditions 104 zoom 24

QuantStudio™ Design and Analysis Desktop Software User Guide 109

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19 October 2018