Determination of Amplification Efficiencies of TaqManGene Expression Assays
Initial testing of 30 assays using a 1-log dilution range (with three concentration points at 10, 5, and 1 ng/μL) and a 3-log dilution range (with four concentration points at 10, 1, 0.1, and 0.01 ng/μL) indicated there were statistical problems with these narrow dilution ranges when measuring amplification efficiencies. Narrow dilution ranges are often used because of the limited amount of cDNA available to do the experiment, especially when quantitating low-copy number transcripts within a cDNA sample.
The use of a broad dynamic range and more data points significantly altered the distribution of amplification efficiency values. The use of a broader log dilution range, however, requires the generation of PCR product or other high concentration template such as a cDNA clone. Therefore, the best protocol for determining amplification efficiency is more difficult to implement.
The initial data indicated that the accuracy of efficiency measurements is extremely sensitive to both the number of data points and the dilution range used in a single experiment. Replicate experiments indicated that the precision of an amplification efficiency measurement increases when the experiment is performed over a broader range of template concentrations, or dilutions.
To further define the variability associated with measuring amplification efficiencies, we tested one assay targeting the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript using either a 2-log (cDNA template) or 5-log (PCR product) dilution series with 5 and 6 concentration points, respectively. M