Amplification Efficiency of TaqMan Gene Expression Assays ( ultiple measurements were performed and...)

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Amplification Efficiency of TaqMan Gene Expression Assays

ultiple measurements were performed and the results are depicted in Figure 2. The 2-log measurements exhibited a much broader range of efficiency values, from 82% to 112% (avg. = 97.6%). The 5-log measurements exhibited much less variation, ranging from 92% to 105% efficiency (avg. = 98.4%). Note that the average efficiency values were not significantly different between the two dilution ranges, and were close to 100%. The data shows how dramatically the range in efficiency measurement can increase (possibly giving very misleading results) over a small dilution range (23 log) versus a larger dilution range (56 log). The increase is due to the fact that the effect of C_T variation (from pipetting error, for example) is minimized when using larger dilution ranges.

The initial data from the first 30 assays and GAPDH allowed us to design the optimal protocol for measuring amplification efficiency of a statistically significant sample of over 750 TaqMan Gene Expression Assays.

The tested assays represented a wide range of oligonucleotide compositions, for example: base composition (26% 79% GC), amplicon length (53 bp186 bp), and secondary structural features (free energy 0 to -19.36 Kcal/mole). Results from sample assays are shown in Figure 3. Our assay design pipeline ensures that the amplicon sizes are the shortest possible, and avoids designs that have larger amplicon sizes. The assays tested had amplification efficiency values ranging from 90% to 110% with very few (four) outliers. Repeat testing of these outliers resulted in values in the 90% to 110% range. The distribution of efficiency is shown in Figure 4. The distribution approximates a normal distribution with a mean of 98.73%.

Determination of Fold Change Fold change in transcript levels for target templates present in the Stratagene Universal Reference RNA dilution series were determined using a spiked non-human transcript (NHT) as an i
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