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) is 0.99 or greater. The closer the slope of this straight line is to -3.32, the closer the amplification efficiency is to 100 percent.
The amplification efficiency = [10(-1/slope)] - 1
Alternatively, a number of data analysis models have been developed that enable the calculation of PCR amplification efficiencies from individual amplification plots, without the use of standard curves. These include the Data Analysis for Real-time PCR (DART-PCR), LinReg, and the Real-time PCR Miner algorithms. Because these methods do not require the generation of standard curves, they are well suited for large scale experiments