2 march 06 Seminar on RT-PCR. About Real-time PCR. Aurélie OLIVIER Université Catholique de Louvain Unité de pharmacologie cellulaire et moléculaire

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1 2 march 06 Seminar on RT-PCR About Real-time PCR Aurélie OLIVIER Université Catholique de Louvain Unité de pharmacologie cellulaire et moléculaire

2 Target DNA PCR Applications: Gene Plasmide, phage Diagnostic 5' 3' 3' 5' Denaturation of target DNA (94 C - 96 C) Annealing of the oligonucleotide primers ( 50 C 60 C) No information about gene expression 5' 5' Synthesis of target DNA (70 C 75 C) cycles

3 Real-Time PCR Quantitation of the starting amount of nucleic acid through the use of appropriate fluorescent detection strategies Applications: Measurements of viral load Gene expression studies Clinical diagnostics Pathogen detection

4 Probe vs. fluorescent dye Polymerization Forward primer Probe R = Reporter Q = Quencher Probe Fluorescent dye SYBR Green I Reverse primer Strand displacement 5' 3' 3' 5' Cleavage 5' 5' Polymerization Quantitation >> mesuring the increase in fluorescence during the exponential phase of PCR

5 Probe vs. fluorescent dye When to use it? Probe High sensitivity Detection of several target in one sample (multiplex) Expensive (~ 200 euro) - Need a very high specificity / cannot find primers that are specific - A lot of reactions looking for the same thing - Sample for Southern blot SYBR Green Inexpensive (~ 2 euro/tube) Less sensitivity (primers dimers, wrong hybridization, ) - Low cost experiment - Looking for different gene expression in a small amount of sample

6 Factors affecting Real-Time PCR 1. General laboratory practices Clean bench coat, gloves, calibrated pipets, 2. Reaction components and conditions Salt composition of the buffer, annealing t, time of extension, 3. Template and primer design

7 Template DNA Step 0: Sampling Step 1: pure bacterial / eucaryotic cell culture Selective media, incubation Extraction kit Step 2: RNA/DNA extraction DNase I treatment Step 3: RNA purification Step 4:absence of DNA contamination Step 4: cdna synthesis PCR Reverse transcription Amplification and analysis Step 5: Real Time PCR

8 Template DNA : Reverse Transcription mrna :~5% rrna : 80% trna :10-15% 15% Reverse transcription kit (Promega): MgCl2 25mM + RT buffer 10x + dntp Mix 10 mm + Rnasin ribonuclease inhibitor ibitor + AMV reverse transcriptase + Random primers + Nuclease free water + RNA Use the recommended amount of RNA!

9 Template DNA : Reverse Transcription Random primers High expression mrna Low expression mrna Less RNA Recommended amount of RNA More RNA

10 Primer design for RT-PCR 1. Target sequence 2. 2 nd structure analysis 3. Think small! 4. Primers design 5. Test in real life conditions

11 Primer design 1. Target sequence Blast in data bank

14 Primer design nd structure analysis Denaturation step (94 c 96 C) >> ssdna Annealing step (50 C 60 C) >> 2 nd structure Impede primers annealing Prevent complete product extension by the polymerase Select primers that don't anneal on a hairpin region

15 Primer design 3. Tink small! 75 pb pb Real-time in 2 step (no extension step) Taq polymerase elongates only a few bp: Polymerase

16 Primer design 4. Primer design Primers that don't anneal on a 2nd structure Annealing temperature above the melting temperature (Tm) for any template secondary structures Amplified target of pb GC content of 50 60% Avoid repeats (>4) of single bases No 3' complementarity (avoid primer-dimer formation) 5' Primer F 3' 3' 5' Primer R 5' ~ 50 pb 5'

17 Primer design 5. Test in real life conditions Gradient Selection of the optimal t Quantitation: Standard and house keeping gene Melt curve Identification of non specific products

18 Primer design 5. Test in real life conditions

19 Gradient A = 59 C B = 58.3 C C = 57.1 C D = 55.2 C E = 52.7 C F = 51.0 C G = 49.8 C H = 49 C Optimal Tm = 57.1 C

20 Standard & house keeping gene Standard : dilution of a template DNA Allows quantification of samples DNA Ex: 10 8 to 10 3 copies Ech. Threshold Log Starting Starting SQ Cycle (Ct) Quantity Quantity (SQ) Mean SI ARN e8 B E E+08 SI ARN e8 B E E+08 hla B E E+09 hlga B E E+07 hlgc B E E+08 16s B E E+09

21 Standard & house keeping gene Same gene than the target gene! Same PCR efficiency Synthesis of standard: size of amplified fragment buffer composition Standard cdna Buffer composition Target gene Target gene

22 Standard & house keeping gene House keeping gene : Gene with a constant expression Normalization of target gene expression >> comparison of different strains / conditions Ex.: rrna16s, hexa, rpsl,,

23 Melt curve Identification of non specific products Denaturation t for PCR product Expected melt curve Primer - dimer

24 Melt curve Identification of non specific products 2 nd product GC-rich sequence

25 Tips for Real-time PCR ( ) 06) Primer design: 1. Target sequence - blast nd structure analysis 3. Think small! pb 4. Primers design 5. Test in real life conditions

Technical Review. Real time PCR

Technical Review. Real time PCR Technical Review Real time PCR Normal PCR: Analyze with agarose gel Normal PCR vs Real time PCR Real-time PCR, also known as quantitative PCR (qpcr) or kinetic PCR Key feature: Used to amplify and simultaneously