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Improving RNA Amplification

s. double round amplification [7, 8]. While possible, interpretation of expression profiles between samples that have been processed differently (single vs. double round amplification) is more problematic. Nevertheless, we attempted to measure the difference observed as the input levels are lowered to 10 ng of total RNA (approximately 1000 cell equivalents).

This experiment consisted of two variables: RNA input (1000, 100, and 10 ng), and amplification protocol (MessageAmp II vs. Affymetrix). For this analysis, both the 100 and 10 ng samples underwent two rounds of amplification.

Note that while the goal of the study was to compare array data generated after two rounds of amplification, MessageAmp II yields from one round of amplification with 100 ng inputs was actually sufficient for array hybridization. In this case, a single round of amplification would be preferred, as it would obviously improve correlation.

Again, we used 1000 ng input RNA as the standard to which we compared the other RNA input data (Figure 9A). Scatter plots of the log2 (expression) for both 100 and 10 ng RNA versus 1000 ng (within a protocol) is shown in Figure 9B. Obviously the 10 ng has much lower fidelity (correlation) with the standard for both MessageAmp II and Affymetrix.

Figure 9. (A) Correlation and Standard Deviation of Differences for RNA Input. Correlation and Standard Deviation are presented for differences in the brain samples, when comparing in
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