An Introduction to qRT-PCR Assays Using Sybr Green (Using SDS ...

that triplicates contain exactly the same reaction components – anydifferences between them are due to technical factors and assay noise,allowing a measure of intra-assay variation to be calculated.8. Data AnalysisThere are a number of approaches to data analysis, many requiring the use ofadditional ‘known’ samples in the form of a standard curve.In brief:2 - ∆∆ CTmethodThis is based upon an assumption of perfect doubling of PCR product percycle and will give a reasonable estimate of expression. An R0 value can besimply calculated for each sample:R0 = Threshold value x (2 -CT value )Eg: if threshold is 0.13 and a sample has a CT value of 21.65, then the R0 valuewill be 0.13 x (2^-21.65) = 3.95 x 10 -8 . The R0 values for target gene samples arethen divided by internal control gene R0 values, to give a gene expressionratio for each sample. The mean and standard deviation may then beexpressed relative to the mean of the experimental control group.Efficiency from Standard CurvesThe use of serial dilutions of cDNA (usually tenfold) can be used to moreaccurately determine efficiency for each primer set. This can also be done forcopy number standard curves, and using this efficiency the actual fold changeand variance achieved is mathematically identical to that produced fromusing copy numbers (see Peirson et al., 2003).Plot the CT of standards (y-axis) against log copy number or cDNAconcentration (will be negative for dilutions – eg: 100 fold dilution of cDNAgives concentration of 0.01, or –2 log units). Fit a linear regression trend-lineto give the slope and fit. The correlation coefficient should be greater than0.95 to ensure accurate standards.The slope of the standard curve can be used to calculate efficiency:Applied efficiency = 10 (-1/slope)So a perfect amplifcation rate of 2 will have a standard curve slope of –3.32.Note: in many publications efficiency is referred to as a reaction rate, and isthus on a scale of 0 (no reaction) to 1 (maximum reaction). Using applied