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QRT-PCR: Quantitative Reverse Transcription PCR

This document discusses quantitative reverse transcription PCR (qRT-PCR). It describes the basic protocol which includes RNA extraction, reverse transcription, amplification, and quantification. Some advantages of qRT-PCR are that it allows analysis of samples with differing target abundance, has little inter-assay variation, and is quantitative. Optimization of qRT-PCR involves choosing the target gene, detection method, oligonucleotides/primers, sample material/processing, and quantification strategy. Primer design considerations and validation of the assay are also discussed.

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Madel Tutor Chaturvedi
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106 views19 pages

QRT-PCR: Quantitative Reverse Transcription PCR

This document discusses quantitative reverse transcription PCR (qRT-PCR). It describes the basic protocol which includes RNA extraction, reverse transcription, amplification, and quantification. Some advantages of qRT-PCR are that it allows analysis of samples with differing target abundance, has little inter-assay variation, and is quantitative. Optimization of qRT-PCR involves choosing the target gene, detection method, oligonucleotides/primers, sample material/processing, and quantification strategy. Primer design considerations and validation of the assay are also discussed.

Uploaded by

Madel Tutor Chaturvedi
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
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qRT-PCR

Quantitative Reverse Transcription PCR

Basic protocol

RNA
Extraction

Reverse
Transcription

Amplification

Quantification

advantages
Combination of DNA amplification and
detection obviates requirements for postPCR processing
Allows analysis of samples differing in
target abundance
Little inter-assay variation
Quantitative rather than qualitative

optimization
Choice of Method
Choice of target gene
Choice of detection method
Choice of oligonucleotides and primers
Choice of sample material and sample
processing
Quantification strategies

optimization
Choice of Method
Choice of target gene
Choice of detection method
Choice of oligonucleotides and primers
Choice of sample material and sample
processing
Quantification strategies

optimization
Choice of Method
Choice of target gene
Choice of detection method
Choice of oligonucleotides and primers
Choice of sample material and sample
processing
Quantification strategies

Detection methods

amplification
plot

Alpha tubulin II
Std dilution: 5-fold

Standard curve
Slope: -3.819
Efficiency: 98.6

Melt curve
Presence of
multiple species

optimization
Choice of Method
Choice of target gene
Choice of detection method
Choice of probes and primers
Choice of sample material and sample
processing
Quantification strategies

Primer design

Length: 18-24 nt
Tm: 58-60OC
GC content: 40-60%
Amplicon: 100-150 bp
Avoid secondary structures
Avoid runs of 4 or more of one base or dinucleotide
repeats
Avoid intra- and inter- primer homology

Primer blast results

Beacon designer results

NetPrimer Results

optimization
Choice of Method
Choice of target gene
Choice of detection method
Choice of oligonucleotides and primers
Choice of sample material and sample
processing
Quantification strategies

optimization
Choice of Method
Choice of target gene
Choice of detection method
Choice of oligonucleotides and primers
Choice of sample material and sample
processing
Quantification strategies

optimization
Validation
Verification of design of oligonucleotides
Verification of amplification
Optimization of reaction conditions
PCR characteristics
Analytical and clinical verification
Internal quality control
Proficiency testing

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