TECHNICAL MANUAL

GoTaq® 1-Step RT-qPCR System

Instructions for Use of Product
A6020

Revised 8/23
TM355
 GoTaq® 1-Step RT-qPCR System
All technical literature is available at: www.promega.com/protocols/
Visit the web site to verify that you are using the most current version of this Technical Manual.
E-mail Promega Technical Services if you have questions on use of this system: [email protected]

1. Description.............................................................................................................................................1

2. Product Components and Storage Conditions...............................................................................................2

3. General Considerations.............................................................................................................................3
3.A. Preventing Contamination................................................................................................................3
3.B. qPCR Primers.................................................................................................................................3
3.C. RNA Template................................................................................................................................3
3.D. BRYT Green® Dye............................................................................................................................4
3.E. CXR Reference Dye and Instrument Considerations..............................................................................4

4. GoTaq® 1-Step RT-qPCR Protocol...............................................................................................................5

4.A. Optional: Adding CXR Reference Dye to the GoTaq® qPCR Master Mix.....................................................5
4.B. Assembling the GoTaq® 1-Step RT-qPCR Reaction Mix..........................................................................5

5. Thermal Cycling......................................................................................................................................6

6. General References for qPCR.....................................................................................................................7

7. Related Products.....................................................................................................................................7

8. Summary of Changes...............................................................................................................................8

1. Description
GoTaq® 1-Step RT-qPCR System(a) combines GoScript™ Reverse Transcriptase and GoTaq® qPCR Master Mix in a
single-step real-time amplification reaction. The system, which is optimized for RT-qPCR, contains a proprietary
fluorescent DNA-binding dye, BRYT Green® Dye. The system enables detection of RNA expression levels using a one-step
RT-qPCR method, combining GoScript™ Reverse Transcriptase and GoTaq® qPCR Master Mix in a single-step real-time
amplification reaction. An overview of the protocol is shown in Figure 1.
The GoScript™ RT Mix for 1-Step RT-qPCR (50X) includes optimized amounts of GoScript™ Reverse Transcriptase, RNasin®
Plus RNase Inhibitor and additives to enhance single-step reactions.
The GoTaq® qPCR Master Mix is a simple-to-use, stabilized 2X formulation that includes all components for qPCR except
template, primers and water. This formulation, which includes a proprietary dsDNA-binding dye, a low level of
carboxy-X-rhodamine (CXR) reference dye (identical to ROX™ dye), GoTaq® Hot Start Polymerase, MgCl2, dNTPs and a
proprietary reaction buffer, produces optimal results in qPCR experiments. A separate tube of CXR Reference Dye is
included for use with instruments that require a higher level of reference dye than that in the GoTaq® qPCR Master Mix.

Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 1
www.promega.com TM355 · Revised 8/23
1. Description (continued)

Prepare the GoTaq® 1-Step

RT-qPCR Reaction Mix by
combining GoTaq® qPCR
Master Mix, GoScript™ RT Mix,
water, primers and template.

Assemble reaction.

Perform RT-qPCR.

Analyze amplification, dissociation

and standard curve data.

10093TA
Figure 1. Overview of the GoTaq® 1-Step RT-qPCR protocol.

2. Product Components and Storage Conditions

PRODUCT SIZE C A T. #

GoTaq® 1-Step RT-qPCR Master Mix 5ml A6020

For research use only. Not for use in diagnostic procedures. Each system contains sufficient reagents for 500 × 20µl reactions. Includes:
• 5 × 1ml GoTaq® qPCR Master Mix, 2X
• 225µl GoScript™ RT Mix for 1-Step RT-qPCR
• 200µl CXR Reference Dye, 30µM
• 750µl MgCl2, 25mM
• 2 × 13ml Nuclease-Free Water

Storage Conditions: Store all components between –30°C to –10°C. Protect components from light at all times. Thaw the
GoScript™ RT Mix for 1-Step RT-qPCR on ice and mix until no visible precipitate is present. Store the buffer on ice after
thawing. For best results, mix thawed solution gently to minimize aeration and foaming, and keep on ice during use. For
short-term storage and frequent use, the GoTaq® qPCR Master Mix can be stored at +2°C to +10°C for up to 3 months if
protected from light.

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TM355 · Revised 8/23 www.promega.com
 Available Separately

PRODUCT SIZE C A T. #

CXR Reference Dye, 30µM* 100µl C5411

Nuclease-Free Water 50ml P1193
*For research use only. Not for use in diagnostic procedures.

3. General Considerations

3.A. Preventing Contamination

We recommend the following precautions to prevent contamination:
• Use designated work areas and pipettes for pre- and post-amplification steps to minimize the potential for
cross contamination between samples and prevent carryover of nucleic acids from one experiment to the next.
• Wear gloves and change them often.
• Do not open the reaction plate or strip wells after amplification is complete. Opening the reaction plate or strip
wells increases the risk of contaminating subsequent reactions with the amplified product.
• Use aerosol-resistant pipette tips.

3.B. qPCR Primers

Optimize the primer concentrations for each primer combination. Primer concentrations can range from 50nM to 300nM;
perform titrations to ensure optimal results. As a general rule, a concentration of 200nM for each PCR primer is a
recommended starting point.
We recommend preparing and storing PCR primers as 20X solutions.

3.C. RNA Template

The amount of RNA required to detect the target of interest depends on several factors, primarily the abundance of that
RNA target in each sample. As a starting point to detect RNA at unknown expression levels, we recommend using 100ng
of total RNA template per reaction. A high-copy-number RNA transcript may be detected in as little as 500fg, while a
low-copy-number RNA transcript may require more than 100ng. Up to 100ng of RNA can be used in each reaction.
For optimal results, the RNA template should be purified to remove genomic DNA contamination. This is particularly
important when using amplification targets within a single exon to avoid amplifying any contaminating genomic DNA.

Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 3
www.promega.com TM355 · Revised 8/23
3.D. BRYT Green® Dye
The BRYT Green® Dye in the GoTaq® qPCR Master Mix has spectral properties similar to those of SYBR® Green I: excitation
at 493nm and emission at 530nm. Use the instrument optical settings established for SYBR® Green I assays with GoTaq®
qPCR Master Mix.

3.E. CXR Reference Dye and Instrument Considerations

The GoTaq® qPCR Master Mix contains a reference dye, carboxy-X-rhodamine (CXR), which is identical to ROX™ and
allows GoTaq® qPCR Master Mix to be used directly on most instruments that perform passive reference normalization,
e.g., from Applied Biosystems. A separate tube of CXR Reference Dye is included with the GoTaq® qPCR Master Mix for
users of instruments requiring a high concentration of reference dye (e.g., ABI 7900). The supplemental CXR Reference
Dye is is provided at a concentration of 30μM.
If you are unsure if your instrument was designed to use no, low or high amounts of ROX™ reference dye for normalization,
contact your instrument vendor.
Recommendations for common instruments are listed below. Directions for setting up qPCRs with supplemental CXR
Reference Dye are included in Section 4.
Instruments That Do Not Require Supplemental Reference Dye
• Applied Biosystems 7500 and 7500 FAST Real-Time PCR System
• Bio-Rad CFX96 Real-Time PCR Detection System
• Bio-Rad/MJ Research Chromo4™ Real-Time Detector
• Eppendorf Mastercycler® ep realplex Real-Time PCR System
• Roche LightCycler® 480 Real-Time PCR System
• Stratagene Mx3000P® and Mx3005P® Real-Time PCR Systems
• Stratagene Mx4000® Multiplex Quantitative PCR System
• Bio-Rad iCycler iQ® and iQ®5 Real-Time PCR Detection Systems
• Applied Biosystems ViiA® 7 Real-Time PCR System
• Applied Biosystems QuantStudio® Real Time PCR Systems
Instruments That Require High Levels (300nM) of Reference Dye
• Applied Biosystems 7300 and 7900HT Real-Time PCR System
• Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems

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TM355 · Revised 8/23 www.promega.com
 4. GoTaq® 1-Step RT-qPCR Protocol

Materials to Be Supplied by the User

• real-time PCR instrument and related equipment (i.e., optical-grade PCR plates and appropriate plate covers)
• sterile, aerosol-resistant pipette tips
• nuclease-free pipettors dedicated to pre-amplification work
• RNA template
• qPCR primers

4.A. Optional: Adding CXR Reference Dye to the GoTaq® qPCR Master Mix
Some real-time PCR instruments require higher levels of CXR Reference Dye; see Section 3.E. For high reference dye
instruments, add CXR Reference Dye to achieve a high dye concentration (500nM), as follows:
1. Thaw the GoTaq® qPCR Master Mix. Do not thaw the master mix at temperatures above room temperature.
2. Vortex the GoTaq® qPCR Master Mix for 3–5 seconds to mix.
3. When using an instrument designated as a high reference dye instrument, add 0.33µl per 20µl reaction for a final
concentration of 500nM.
4. Vortex for 3–5 seconds to mix.

4.B. Assembling the GoTaq® 1-Step RT-qPCR Reaction Mix

The final reaction volume in this protocol is 20µl. The volumes given here may be scaled for larger or smaller reaction
volumes.
1. Thaw the GoTaq® qPCR Master Mix and Nuclease-Free Water. Do not thaw the GoTaq® qPCR Master Mix at elevated
temperatures (i.e., above room temperature).
2. Vortex the GoTaq® qPCR Master Mix for 3–5 seconds to mix. Vortex at low speed to avoid aeration.
3. Determine the number of reactions to be set up, including negative control reactions. Add 1 or 2 reactions to this
number to compensate for pipetting error. While this approach does require using a small amount of extra reagent,
it ensures that you will have enough reaction mix for all samples.

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www.promega.com TM355 · Revised 8/23
4.B. Assembling the GoTaq® 1-Step RT-qPCR Reaction Mix (continued)
4. Prepare the reaction mix (minus RNA template) by combining the GoTaq® qPCR Master Mix, GoScript™ RT Mix, PCR
primers and Nuclease-Free Water as described below. The RNA template is added in Step 6. Vortex briefly to mix.

Component Volume Final Concentration

GoTaq qPCR Master Mix, 2X
®
10µl 1X
GoScript™ RT Mix for 1-Step RT-qPCR (50X) 0.4µl 1X
Forward Primer (20X) ____µl 50–300nM
Reverse Primer (20X) ____µl 50–300nM
CXR Reference Dye (optional) 0.33µl/20µl reaction 500nM
Nuclease-Free Water to a final volume of 20µl
Note: The primer concentrations should be optimized for each primer combination.
5. Add the appropriate volume of reaction mix to each PCR tube or well of an optical-grade PCR plate.
6. Add the RNA template (or water for the no-template control reactions) to the appropriate wells of the reaction plate.
7. Seal the tubes or optical plate, and centrifuge briefly to collect the contents of the wells at the bottom. Protect from
extended light exposure or elevated temperatures. The samples are ready for thermal cycling.

5. Thermal Cycling
The cycling parameters below are offered as a guideline and may be modified as necessary for optimal results.
Standard Cycling Conditions

Step Cycles Temperature Time

Reverse transcription 1 ≥37°C 15 minutes
Reverse transcriptase inactivation and 1 95°C 10 minutes
GoTaq® DNA Polymerase activation
Denaturation 95°C 10 seconds
Annealing and data collection 40 60°C 30 seconds
Extension 72°C 30 seconds
Use the instrument optical settings established for SYBR® Green I assays with GoTaq® qPCR Master Mix.

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TM355 · Revised 8/23 www.promega.com
 6. General References for qPCR
1. Bustin, S.A. et al. (2009) The MIQE guidelines: Minimum information for publication of quantitative real-time PCR
experiments. Clin. Chem. 55, 611–22.
2. Dorak, M.T (2009) Glossary of real-time PCR terms. This can be viewed online at:
www.dorak.info/genetics/glosrt.html
3. Fleige, S. and Pfaffl, M.W. (2006) RNA integrity and the effect on the real-time qRT-PCR performance. Mol. Aspects
Med. 27, 126–39.
4. Lefever, S. et al. (2009) RDML: Structured language and reporting guidelines for real-time quantitative PCR data
Nucleic Acids Res. 37, 2065–9.
5. Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene expression data using real-time quantitative PCR
and the 2– ΔΔCT Method. Methods 25, 402–8.

7. Related Products

Real-Time PCR

Product Size Cat.#

GoTaq® qPCR Master Mix* 5ml A6001
25ml A6002
GoTaq® 2-Step RT-qPCR System* 5ml A6010
GoTaq Probe qPCR Master Mix
®
2ml A6101
10ml A6102
GoTaq Probe 1-Step RT-qPCR System
®
2ml A6120
12.5ml A6121
GoTaq Probe 2-Step RT-qPCR System
®
2ml A6110

RNA Purification, Manual Systems

Product Size Cat.#

ReliaPrep™ RNA Cell Miniprep System 10 preps Z6010
ReliaPrep™ RNA Tissue Miniprep System 10 preps Z6110
ReliaPrep™ FFPE Total RNA Miniprep System 10 reactions Z1001
SV Total RNA Isolation System 10 preps Z3101
PureYield™ RNA Midiprep System 10 preps Z3740
Additional sizes are available.

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www.promega.com TM355 · Revised 8/23
7. Related Products (continued)

Manual or Automated RNA Purification

Product Size Cat.#

SV 96 Total RNA Isolation System 1 × 96 each Z3500
5 × 96 each Z3505
Vac-Man® 96 Vacuum Manifold 1 each A2291

Automated RNA Purification

Product Size Cat.#

Maxwell 16 LEV simplyRNA Cells Kit
®
48 preps AS1270
Maxwell® 16 LEV simplyRNA Tissue Kit 48 preps AS1280
MagneSil Total RNA mini-Isolation System
®
4 plate Z3351

Accessories

Product Size Cat.#

GoScript™ Reverse Transcription System 50 reactions A5000
100 reactions A5001
GoScript™ Reverse Transcriptase 100 reactions A5003
500 reactions A5004
RNasin® Plus RNase Inhibitor 2,500u N2611
10,000u N2615
Recombinant RNasin® Ribonuclease Inhibitor 2,500u N2511
Nuclease-Free Water 50ml P1193

8. Summary of Changes
The following changes were made to the 8/23 revision of this document:
1. Updated Sections 2 and 7.
2. Replaced cover image and document font.
3. Updated patent statements.
4. Made minor text edits.

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TM355 · Revised 8/23 www.promega.com
 U.S. Pat. Nos. 8,598,198 and 9,206,474 and other patents and patents pending.
(a)

© 2011–2023 Promega Corporation. All Rights Reserved.

BRYT Green, GoTaq, MagneSil, Maxwell, RNasin and Vac-Man are registered trademarks of Promega Corporation. GoScript, PureYield and ReliaPrep are
trademarks of Promega Corporation.

Chromo4 is a trademark of Bio-Rad Laboratories. iCycler and iCycler iQ are registered trademarks of Bio-Rad Laboratories, Inc. LightCycler is a registered
trademark of Roche Diagnostics, GmbH. Mastercycler is a registered trademark of Eppendorf-Netheler-Hinz GmbH. Mx3000P, Mx3005P and Mx4000 are
registered trademarks of Agilent Technologies, Inc. QuantStudio and ViiA are registered trademarks of Life Technologies, Inc. ROX, StepOne and StepOnePlus are
trademarks of Applera Corporation. SYBR is a registered trademark of Molecular Probes, Inc.

Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more information.

All prices and specifications are subject to change without prior notice.

Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date information on
Promega products.

Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 9
www.promega.com TM355 · Revised 8/23