real time pcr applications-training-june 2010

Real Time PCR & its applications

Dinesh Kumar

M.Sc. Biotech(BHU), Ph. D. Biotech(BHU), PDF(USA)

Senior Scientist (Animal Biotechnology)Genes & Genetic Resources Molecular Analysis Lab

National Bureau of Animal Genetic ResourcesKarnal-132001

E-mail: [email protected], +91-94161-11753(SMS only)

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Dinesh-NBAGR-Real time Time PCR Applications-

Summer Training-01-June-2010

Why to measure gene expression?

•Chargoff's law two laws•Traits vary•Structural variants of gene•Functional variants of gene•Structural genomics to functional genomics

Why to quantify mRNA?

Gene expression studies

Measurement of mRNA Cell- 100 mRNA of gene A Extract- Northern blotting-densiometry Extract-cDNA-template-PCR-threshold Accuracy in number? 40, 50, 60 molecules Accuracy loss

Extraction Transfer/blotting Hubridisation/washing Signal/noise Reading/manual error

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How to maximize the accuracy?

Use of PCR Measure the threshold (Ct) Compare with house keeping genes. Relative gene expression. Straight line equation(range) PCR efficiency 100% How to do all these?

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How to quantify mRNA?

Northern blotting: In situ hybridization: RNAse protection assays: RT-PCR

Gene XXXXx XXXX X

GAPDH

+/

+ +/

+

1

2

3

kb

0.0

0.5

1.0

1.5

2.0

2.5

Thymus Spleen Lymph Kidney

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How real time PCR machine works?

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How the fluorescence monitoring is done in real time PCR machine?

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Advantages of qPCR 1. Wide dynamic range of quantification (7–8 log

decades).2. High technical sensitivity (< 5 copies).3. High precision (< 2% CV of CT values).4. No post-PCR steps like running of gel or sequencing

etc.5. Non-specific amplification can be detected by melt

curve analysis of PCR products.6. Minimized risk of cross contamination.7. High throughput.8. Multiplex approach possible.9. Saves lot of time.

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Limitations of qPCR

1. PCR product increases exponentially.2. Variation increases with cycle number.3. Increased variation after transformation

to linear values.4. Overlap of emission spectra.5. Maximal four simultaneous reactions

limit the bacterial diagnostics.6. Increased risk of false negative results

where parasite genome changes very fast.

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Primers for RT

• Specific primers: decreases background priming:

• Random hexamers: maximizes the number of mRNA molecules thus often overestimate mRNA copy numbers.

• Oligo-dT primers: maximizes the number of mRNA molecules

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Quantification of mRNA- in vitro

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RNA-poor quality

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Primer/Probe designing for qPCR

Primer ExpressMolecular Beacon-3Primer 3.0OLIGOTm calculatorm-fold

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Common qPCR Instruments

1. ABI Prism 7700, 7000 (Perkin-Elmer–Applied Biosystems, Foster City, CA, USA)

2. Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany)

3. Biorad Instruments- i cycler4. Corbett Research-Rotor Gene5. Opticon-II, Chromo 4 (MJ Research)6. Stratagene (Mx4000)7. Techne-Cephid-Smartcycler

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Basic chemistries used in qPCR

1. DNA-binding dyes

SG (S) will not bind to ssDNA and the intensity of fluorescent signal is low

b) SG (S) binds to dsDNA the fluorescent signal intensity (E) increases

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Advantages of DNA-binding dyes

1. This method obviates the need for target-specific fluorescent probes, but its specificity is determined entirely by its primers.

2. Simple and fast.3. Melt curve analysis ensures

specificity of amplified PCR products.

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Melt curve analysis

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Limitations of DNA-binding dyes any double-stranded DNA

generates fluorescence amplification of a longer product

will generate more signal than a shorter one.

amplification efficiencies different- inaccurate result.

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2. Molecular beacons

The hairpin structure causes the MB to fold when not hybridized, bringing quencher and fluorophore dyes in close proximity causing quenching of fluorescence. When hybridized, the fluorophore and quencher are separated resulting in increased fluorescence.Advantages of Molecular Beacons:specificity non-PCR amplification assays Limitations

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Limitations of molecular beacons

design of the hybridization probe. interfere with the hybridization

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Hybridisation probes fluorescence resonance energy

transfer (FRET)

Resonance energy (E) transfer is low when the probes are not hybridised.

Hybridisation of the probes brings the donor (D) and acceptor (A) fluorophores into close proximity resulting in increased resonance energy transfer.

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Advantages of Hybridization probes:

increases specificity probes are not hydrolyzed,

fluorescence is reversible :allows the generation of melting curves.

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Hydrolysis probes

Energy (E) emitted by the donor (D) fluorophore is absorbed/quenched by the acceptor (A) fluorophore.

The polymerase exonuclease activity separates the D fluorophore from the A fluorophore by hydrolysis resulting in an increase in fluorescent signal.

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Limitations of Hydrolysis probes

reduces the processivity of the polymerases

less efficient when mutation-specific RT-PCR

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Amplicon design

< 100 bp, no longer than 80 bp are found to be ideal

as long as 400 bp minimum 63 bp . Primer and probe design

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Primer & probe designing Primers

15-30 bp in length G/C content of 20-80% Avoid primer dimers. The Tm should be within 2°C Purify by gel electrophoresis or HPLC Optimize concentrations by performing matrix of 50nM, 300nM and

900nM fo the forward and reverse primers. This range allows the matching of the experimental Tm of the primers by +/- 2 °

Probes 20-30 bp in length G/C content of 20-80% Tm 7-10 ° higher than primers To maximize signal or reporter vary the probe concentration between

5-400nM

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Data analysis in qPCR What is the Ct-value?

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Amplification Plots

Kumar SYBR Green, 12-18-2003, 10Hr 00Min.mxp.txt

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Dissociation Curve


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Standard Curve


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What are the reaction efficiency, amplification and slope and how is it calculated?

smaller the Ct value (y intercept) in the regression equation, the greater the sensitivity of the RT-PCR reaction.

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Amplification Plots


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Factors affecting reproducibility of Ct

1. When work with low copy number.2. Short amplicons have greater

reproducibility.3. It is always better to use replicates

for greater accuracy.

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Why and how to Normalize expression data? any variation in the amount of starting

material a cellular RNA that serves as an internal

reference against which other RNA values can be normalized.

housekeeping genes (HKGs) GAPDH , -actin , rRNA H3 and cyclophilin, tubulin, micro-

globulin etc

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Absolute Quantification

C (cRNA} µg/lN (molecules perµl) = __________________ X 182·5×1013

K (fragment size /bp) Where, N= gene molecules per µl, C = concentration of the cRNA inµg/l, K= fragment size or bp, 182·5×1013 Avagadro constant.

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Relative and Comparative Quantification relative to an active reference

control (normalizer or house keeping genes)

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Relative and Comparative Quantification

Advantages 1. No standard

curve is required.

2. More wells available for samples.

Disadvantages 1. More optimization

and validation required

2. If dynamic range of HKG and target genes are not of similar magnitude it needs correction factors.

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Mutation/allele detection

A single base mismatch under the probe

Decreases the Tm by as little as 3 0C for G–T

By 10 0C for A–C mismatches.

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Applications Quantification

Absolute Gene Expression Viral, Fungal, Bacterial

Load Genomic Mitochondrial DNAs Quality Control

Relative and Comparative

Gene expression Microarray validation

Detection Sequence Detection

Allele discrimination SNP analysis Zygosity testing Zoonotic agents GMO screening Residual disease Splice variants Chromosomal

translocations Methylation

Fluorescence Detection Isothermal signal

amplification Plate reader functionality

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Multiplexing 4 channel machine Single PCR tube-data of 4 different

genes Problems Advantages

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Ten most common Real-Time qPCR Pitfalls

Poor primer and probe design Poor quality RNA Not using “master mixes” Introducing cross contamination Not using a “-RT”control Using and inappropriate normalization control Melt curve analysis not done when using SYBR Green Baseline and threshold not set correctly Efficiency of the reaction is poor Using an inappropriate range for standard curve

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Reaction set up

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Real time monitoring

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Dinesh Kumar

Scientist (Animal Biotechnology)Small Ruminant Molecular Genetics Lab

DNA Fingerprinting UnitNational Bureau of Animal Genetic Resources

Karnal-132001E-mail:[email protected], 094161-11753

I am thankful to Dr J.M.Reecy and his group for Making me happen to learn a b c of real time in real spirit!

Thank you to every one here !11/04/23 46



real time pcr applications-training-june 2010

Technology

real time pcr machine

applications dinesh

postpcr steps

curve analysis of pcr

mrna of gene

number of mrna molecules

quantification of mrna

fluorescence amplification