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Introduction to qPCR

The document provides an introduction to quantitative PCR (qPCR), detailing its principles, methods, and applications in molecular biology. It explains the differences between classical PCR and qPCR, including absolute and relative quantification of DNA and gene expression. Additionally, it covers high-resolution melting techniques and compares the use of intercalating dyes and hydrolysis probes in qPCR.

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cernaa.26715
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2 views

Introduction to qPCR

The document provides an introduction to quantitative PCR (qPCR), detailing its principles, methods, and applications in molecular biology. It explains the differences between classical PCR and qPCR, including absolute and relative quantification of DNA and gene expression. Additionally, it covers high-resolution melting techniques and compares the use of intercalating dyes and hydrolysis probes in qPCR.

Uploaded by

cernaa.26715
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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Introduction to

qPCR
JIRI CERVEN, PHD.
Outline:
 1) Classical (end-point) PCR
 2) How does qPCR work?
 A) Intercalating dyes
 B) Hydrolysis probes
 3) What can be measured by qPCR
 A) Absolute quantification
 B) Relative quantification – gene expression
 C) High resolution melting – SnP, mutations, genotyping
End point PCR
 Originally technique to amplify DNA in-vitro; today one of the mostly
used mol-biol. method
 Dr. Kerry Mullis, nobel price 1993
 Principle
End point PCR
1) Amplification (for sequencing
etc.)
2) Lenght differenciantion (for
forensics, mol-biol. systematics
etc.)
3) Parasite detection
4) Fenotypisation
qPCR
 Based on monitoring amplification in the real time based on
measuring of fluorescence
 Two main methods (intercalating dyes and hydrolysis probes)
 Gives information on concentration (quantity) of specific DNA in the
sample
qPCR
What is qPCR good for – absolute quantification

 We can find out exact amount of DNA molecules in sample


 We can detect if a DNA molecule is present in a sample
 We can measure a gene copy number, exact number of genes
 Examples: Detection of pathogens (producers DNA) in vaccines, detection of
parasites/viruses, quantification of uncultivable microorganisms in environmental samples
qPCR – relative quantification
 Most common usage of qPCR – relative quantification of gene expression

• Analysis of specific mRNA level


• We compare, if there is increase
(decrease) of a specific gene after
specific treatment.
• We are not interested in how much mRNA
(protein) is produced, bud if there is a
change in expression.
qPCR – relative quantification
 RNA isolation
 Very sensitive – RNA is very easily degraded!
 Various kits, but most comonly TRIreagent
 Need to control RNA quality – RIN – RNA Integrity Number
qPCR – relative quantification
Reverse transcription
• Using either poly(dT) or
random hexamer
primers

• Useful to use DNAse


prior to cDNA synthesis

• Most expensive part of


qPCR gene expression
evaluation.
qPCR – relative quantification
 Comparing expression of genes in control vs „treatment“
 Treatment: chemical, drug, time, different tissue, different cell type etc.
 We need gene, that is expressed simillarly in control and treated sample – reference gene
 We compare difference in expression between reference and target in control and
treated sample
qPCR – relative quantification
 𝑅 = 2−∆∆𝐶𝑞
 𝑅 = 2−(∆𝐶𝑞 𝑒𝑥𝑝. −∆𝐶𝑞(𝑐𝑜𝑛𝑡.)

 ∆𝐶𝑞 𝑒𝑥𝑝. = 𝐶𝑞 𝑡𝑎𝑟, 𝑒𝑥𝑝. − 𝐶𝑞 𝑟𝑒𝑓, 𝑒𝑥𝑝.


 ∆𝐶𝑞 𝑐𝑜𝑛𝑡. = 𝐶𝑞 𝑡𝑎𝑟, 𝑐𝑜𝑛𝑡. − 𝐶𝑞 𝑟𝑒𝑓, 𝑐𝑜𝑛𝑡.

1E+10
9E+09
8E+09
7E+09
6E+09
5E+09
4E+09
3E+09
2E+09
1E+09
0
25 26 27 28 29 30 31 32 33 34 35

2 1,95 1,9 1,8 Treshold


qPCR – relative quantification

Bar diagram

Heat map
High Resolution Melting
1) First as technique for amplicon purity

Melting profile affected by:


1) GC/AT content
2) Secondary structures
3) Amplicon length
High Resolution Melting - usage

Mutation scanning (SnP)

Genotyping
HRM – real life applications
qPCR – SYBR and probes – pros and cons
 Intercalating dyes:  Hydrolysis Probes
 Pros:  Pros:
 Universal for every reaction  Very specific
 Cheaper  Multiplexing
 Possible HRM detection

 Cons:  Cons:
 Primer-dimers  More expensive
 Not possible to multiplex  More difficult do design primers
 No HRM detection
Digital PCR

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