ÚVOD DO KVANTITATIVNÍ REAL-TIME PCR RT-qPCR • Accuracy, sensitivity, fast results • Monitoring of amplification in real-time RT-PCR • Experimental design • RNA extraction • RNA quality control • Reverse transcription • Primer and amplicon design • qPCR validation • Choice of reference genes • Experimental reproducibility Experimental design • mRNA transcription sensitive to external stimuli- need to minimize • Define – procedures – control groups – type and number of replicates – experimental conditions- minimize variability RNA extraction • From ‘fresh’ material if possible • RNA stored at -80C or in RNA storage solution • Minimize handling time- 10-20 samples • DNAse I treatment RNA quality control • High purity (no contamination) • High integrity (not degraded) • Impurities- PCR inhibition • Purity- protein contamination • OD 260/280 1.8-2.0 no protein • OD 260/230 1.8-2.0 no organic contaminants • RIN>7 • Consistency in purity and integrity-reduction of variability of samples • Immediately follow with procedures, store only cDNA RT • Immediately after isolation- no degradation of RNA from freeze/thaw • Consistent and complete coverage of transcribed regions • Always enter same amount of RNA and same reaction time for all samples • Ctrl – no RT samples (contamination with genomic DNA) – no template control (contamination) • RT Buffer – mix of random primers – RNAse H – RT enzyme- broad dynamic range Primer and amplicon design • Essential for specific and efficient amplification • Target sequences – Unique – 75-100 bp – GC content 50-60% – No secondary structures • Primers – GC content 50-60% – Melting temperature 55-65C – No long stretches of G or C – G or C at the end of primer – Primer blast, MFOLD, experience qPCR validation • Assessed for optimal range of primer annealing temperatures, efficiency, specificity using a standard set of samples • Reaction conditions, buffers, primers optimized • cDNA samples not contaminated • www.bio-rad.com/genomics/pcrsupport qPCR validation • Optimal annealing T for primers- temperature gradient • Analysis of PCR product- melt curve analysis (single sharp peak) • Samples run on gel • NTC necessary (primer –dimer, DNA contamination) PCR efficiency • Measure of rate at which polymerase converts the reagents to amplicon • Maximum increase per cycle is 2-fold – 100% efficiency • Low efficiency – inhibitors of polymerase – high or suboptimal annealing temperature – old/inactive Taq – poorly designed primers – secondary structures Standard curve • 10-fold dilution – 8 points • Broad dynamic range • For each point- in triplicates, get Ct values • Need tight technical replicates • If OK- Ct values separated by 3.32 cycles • Need 90-110% Efficiency • R values- how well data fit on curve • R2>0.985 OK • Will define dynamic range of reaction qPCR • Commercial qPCR kits • Sample volumes- 10-50ul in 96 well plate format • Software analysis – Flexibility in set up info – Group wells – Gene expression analysis – Ability to combine multiple plates Choice of reference genes • Perfect reference gene- no expression changes between samples from various experimental conditions, time points • How to find: – Extract RNA from 1-2 samples from each condition or time point, confirm purity and quality – Normalize concentration, do RT from same volume – Do qPCR from same volume of cDNA – geNorm method to calculate stability (med- gen.ugent.be/genorm/ – Need 3-5 genes Experimental reproducibility • 3 biological and 2 technical replicates • 3 biological replicates- separate and independent experiments • 2 sources of variability: – Biological- differences of organisms, tissues, cell cultures – Technical- pipetting, samples quality... Key steps for qPCR • Appropriate number of biological replicates and control samples • Strict protocols for acquisition, processing and storage • RNA purity and integrity • RT • Proper design of PCR Kvantitativní vztah mezi množstvím PCR produktu (amplikonu) a intenzitou fluorescence • Amplifikační práh detekce (Ct) Real – time detekce amplifikace Plateau fáze Exponenciální amplifikace pod úrovní pozadí Exponenciální fáze „end point“ „real-time“ Threshold cycle „Ct“ – určený na základě hodnoty fluorescence pozadí (backround) a aktuální fluorescence vzorku – kvantitativní výstup pro každý vzorek Real – time detekce amplifikace - Ct Threshold Threshold cyclesBackground Fluorescence Threshold cycle „Ct“ - počáteční množství kopií templátu - definovaný v exponenciální fázi PCR - stejná účinnost PCR ve všech reakcích - účinnost štěpení fluorogenní sondy nebo vazby fluoroforu na DNA - citlivost detekce - čím menší Ct - tím větší počet kopií templátu na začátku reakce Real – time detekce amplifikace - Ct A CB A CB> > - rozdíl 1 Ct – dvojnásobné množství templátu 21 = 2 - kolika cyklům odpovídá odpovídá 10ti násobný rozdíl v množství templátu? (předpokládáme 100% účinnost PCR) 2n = 10 Threshold cycle „Ct“ Real – time detekce amplifikace - Ct 50 100 150 250 300 0 c [ng/µl] Ct 50 28,16 100 27,16 150 26,66 250 26,06 300 25,66 n=3,32 Kontaminace PCR Cross contamination Carry-over contamination PCR 1 PCR 2 PCR 2PCR 1 Vzorek 1 Vzorek 2 Přenos amplikonu do dalších PCR Vzájemná kontaminace vzorků Jak předejít kontaminaci Kontaminace • Správná laboratorní praxe • Plastik v RNA kvalitě • Automatizace × www.millipore.com; www.appliedbiosystems.com Application note: Contamination-pipetting: relative efficiency of filter tips compared to Microman® positive displacement pipette. Nature Methods 3 June 2006