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Improving RNA Amplification

puts of 10, 100, and 1000 ng to the standard (1000 ng). (B) Scatter Plots of Log2 (expression) by RNA amount. Each input is plotted vs the standard of 4 hour IVT using 1.0 g for both MessageAmp II (top row) and GeneChip IVT Labeling Kit (bottom row). The 0.1 g and 0.01 g samples are derived from two rounds of amplification.

As done previously, for comparison we used the standard deviation of the differences between two replicates of the standard (0.15 and 0.17). The results suggests that fidelity decreases with decreasing RNA input, but particularly for the 100 ng concentration, the correlation (and standard deviation of differences) is better for the MessageAmp II protocol than the Affymetrix standard protocol (0.3 vs. 0.55). At 10 ng inputs fidelity is lower. Nevertheless, the signal correlation indicates meaningful data can still be obtained (0.91 vs. 0.93). Again, in some cases two rounds of amplification are not needed (e.g. for 100 ng inputs with the MessageAmp II procedure), and in this case a comparison between the 1000 ng standard and 100 ng single round amplification is significantly more accurate and applicable.


Conclusion
The critical criteria for microarray sample preparation methods are: they must be highly reproducible, conserve the original mRNA profile, and be applicable with a reasonable range of total RNA inputs. While it is important that both amplification and labeling methods conserve the original expression profile, we re
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