Introduction to real-time PCR Erik Lysøe Fusarium, genes, and toxins 2008
Overview Principles, applications and comparison to traditional PCR Pi Primer design key to succesful PCR Quantification strategies, absolute / relative PCR efficiency Useful links and good real-time PCR sources
Principles of real-time PCR Sensitive, specific and reproducible quantitation of nucleic acids Fluorescence is measured every cycle, signal amount of PCR product Detection of signals during early phases of reaction 10000000000 1000000000 100000000 10000000 1000000 100000 10000 1000 100 10 1 Curves rise after a number of cycles that is proportional to the initial amount of DNA template 100 AMOUNT OF DNA 0 5 10 15 20 25 30 35 PCR CYCLE NUMBER
Real-time PCR applications Quantitation of gene expression Pathogen detection GMO detection Viral quantitation Array verification Drug therapy efficacy DNA damage measurement Quality control and assay validation Genotyping
CYCLE NUMBER AMOUNT OF DNA 0 1 1 2 Real-time vs traditional PCR 2 4 End-Point 3 8 4 16 5 32 6 64 7 128 8 256 9 512 10 1,024 11 2,048 12 4,096 13 8,192 14 16,384 15 32,768 16 65,536 17 131,072 18 262,144 19 524,288 20 1,048,576 21 2,097,152 22 4,194,304 23 8,388,608 24 16,777,216 25 33,554,432 26 67,108,864 27 134,217,728 28 268,435,456 29 536,870,912 30 1,073,741,824 31 1,400,000,000 32 1,500,000,000 33 1,550,000,000 34 1,580,000,000
Limitations of end-point PCR Time consuming, non-automated Poor precision, size-based discrimination only Low resolution about 10 fold. Real-Time PCR can detect as little as a two-fold change! Results are not expressed as numbers Ethidium bromide for staining is not very quantitative Low sensitivity, not necessarily related to amount of input DNA Short dynamic range < 2 logs Post PCR processing
Dynamic range 10 8 or more!
Setting threshold
Setting threshold SERIES OF 10-FOLD DILUTIONS As one dilutes the sample, it takes more cycles before the amplification is detectable.
threshold = 300 It is important that the threshold should be in the linear part of the reaction (easier to see in the logarithmic view). The threshold should be high enough that you are sure that reactions cross the line due to amplification rather than noise. Uuse the same threshold for all the samples in the same experiment on the same plate.
SYBR Green binds to dsdna Commonly used in gene expression assays
The TaqMan probe Commonly used in diagnostic assays
Probes: table of comparison
Multiplex l Several targets amplified simultaniously in the same tube Several primers for several templates Several probes Quantification of target gene e and reference e e gene e in one reaction
Primer design key to successful PCR Good primer design saves time and money Advanced applications require even more stringent primer design» Multiplex» Single-cell real-time PCR Primer Express software
Primer design key to successful PCR Physical parameters What affects DNA-DNA interactions? Temperature Base composition o Concentration of primers Time What properties of the PCR are affected? Specificity it Efficiency Sensitivity
Good primer (pair) properties Primers should have 18-24 bases 40-60% G/C Balanced distribution ib i of G/C and A/T bases Avoid runs ( 4) of an identical nucleotide (especially G s) Tm that allows annealing at 55-65 CC No internal secondary structures (hair-pins) The five nt at the 3 end should have no more than two G and/or C bases Primer pairs should have Similar melting temperatures, T m, within 2-3 C No significant complementarity (> 2-3 bp), particularly not in g p y ( p), p y the 3 -ends
SYBR Green dissociation curve (T m ) Primer-dimer
Solutions to primer dimer problem Reduce the formation of PDs by Good primer design (avoid 3 complementarity) Minimal i annealing time Good laboratory practice HotStart TouchDown Reduce the signal from PDs by Measuring fluorescence above the T m of the PDs Use sequence-specific probe
Considerations
Assay validation Factors affecting the results Biological variation Sampling Storage of samples Lysis and RNA extraction RNA /cdna storage Reverse trancription Choice of oligo dt, random hexamers for cdna synthesis Standards Valdiation experiments PCR efficiency Selection of method Endogenous control Statistics i
Quantification strategies t Absolute quantification Standard curve with known (mrna / DNA) 100 % efficient assays may be highly sensitive Relative quantification Compare amount using a reference normalization If using the 2 ΔΔC T (Livak) method or the ΔC T method using a reference gene, it s only ypossible to compare assays with similar or very high PCR efficiency. If PCR efficiency is taken into account (Pfaffl method), more variation in PCR efficiency is tolerated
Absolute quantification Requires the construction of an absolute standard curve for each target The standard curve is based on a serial dilution of a sample with known copy number Ct of each standard sample is plotted against the logarithm of the known concentration The standard curve is then used to estimate concentrations of unknown samples
Standard d curve
Relative quantification Often there are no good standard available Compare amount with reference Housekeeping genes rrna Genomic DNA
Relative quantification The 2 ΔΔC T (Livak) method The ΔC T method using a reference gene The Pfaffl method
The 2 ΔΔC T (Livak) method This method assumes that both target and reference genes are amplified with efficiencies near 100% and within 5% of each other. First, normalize the CT of the target gene to that of the reference (ref) gene, for both the test sample and the calibrator sample: Second, normalize the ΔC T of the test sample to the ΔC T of the calibrator: Finally, calculate the expression ratio:
Continue The 2 ΔΔC T (Livak) method
The ΔC T method using a reference gene The ΔC T method using a reference gene is a variation of the Livak method that is simpler to perform and gives essentially the same results. This method uses the difference between reference and target C T values for each sample.
The Pfaffl ffl Method The 2 ΔΔC T method for calculating relative gene expression is only valid when the amplification efficiencies of the target and reference genes are similar. If the amplification efficiencies of the two amplicons are not the same, an alternative formula must be used to determine the relative expression of the target gene in different samples. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001 May 1;29(9). How to determine E?
PCR Efficiency
PCR Efficiency Large range ( 5 orders of magnitude) Enough replicates ( 3, 4 for fast) All points in linear range, no outliers All samples should be within standard curve range!!
PCR Efficiencyi
Endogenous control (housekeeping gene) The perfect EC reflects the amount of DNA/cDNA per well Sould have constant expression level in all samples wich are used in the study Normalizes for RNA input measurement errors RT efficiency i variations House-keeping genes are often used There is no universal EC
Optimization or not? Taqman assay Each primer: 900 nm Probe: 200 nm Universal assay conditions SYBR Green First shot, but not universal: Each primer 100 nm Optimalization with primer matrix: 50,300,900 for Taqman and 100,200,300 for SYBRGreen Choose pair with lowest Ct value Optimalization with probe: 50,100,150,200 Optimalization with probe: 50,100,150,200 Recommended RN value (fluorescence) 0.8-1
Useful links and real-time PCR sources http://www.tataa.com/ Commercial knowledge center for real-time PCR http://www.gene-quantification.de REST Bestkeeper etc... Real-Time PCR Applications Guide (BioRad) http://www.bio-rad.com/pdf/bulletin_5279b.pdf pdf Real-time PCR tutorial http://pathmicro.med.sc.edu/pcr/realtimemed edu/pcr/realtime-home.htmhtm User Bulletin #2 - ABI PRISM 7700 Sequence Detection System http://docs.appliedbiosystems.com/pebiodocs/04303859.pdf pp p p