What is the standard curve method for qPCR assay data analysis? How is the standard curve method for qPCR assay data analysis performed?
When using the standard curve method, the quantity of each experimental sample is first determined using a standard curve, and is then expressed relative to a calibrator sample.
In order to use this quantification method, prepare five (5) 2-fold, 5-fold, or 10-fold serial dilutions of cDNA template known to express the gene of interest in high abundance. Use each serial dilution in separate real-time reactions, and determine their threshold cycle values.
In a base-10 semi-logarithmic graph, plot the threshold cycle versus the dilution factor and fit the data to a straight line. Confirm that the correlation coefficient (R2) for the line is 0.99 or greater.
This plot is then used as a standard or calibration curve for extrapolating relative expression level information for the same gene of interest in unknown experimental samples. The relative quantification calibration curve result for the gene of interest is normalized to that of a housekeeping gene in the same sample, and then the normalized numbers are compared between samples to get a fold change in expression.
A standard or calibration curve must be generated separately for each gene of interest and each housekeeping gene.
See Critical Factors for Successful Real-Time PCR for additional details.