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Improving RNA Amplification

ns. Both MessageAmp II (top) and Affymetrix standard protocol (bottom) were performed with the identical total RNA (1 g) and equivalent amounts were hybridized on matched lot Human Genome Focus arrays. All IVT reactions were performed for 4 hours. Correlation values are included and indicate a measure of reproducibility with both methods.(B) Correlation and Standard Deviation of Differences for RNA Inputs and IVT Incubation Times Compared to Standard. Two sources of total RNA brain and HeLa S3 cells, were used for amplification, and 4, 6, and 14 hr IVT reaction times were tested. For comparison the same information is provided for replicates of the Affymetrix protocol.


Differential Gene Expression. We also addressed differential gene expression between two different tissue sources (brain and HeLa). To measure the consistency of the MessageAmp II and the Affymetrix standard protocol, we plotted the possible replicates of the ratios for both protocols. Since there are two replicates of input RNA from brain and two from HeLa, for each protocol there are 4 possible ratios: 2 sets of 2 contain unique arrays.We compared the consistency of MessageAmp II and Affymetrix ratio estimates in Figure 6. There is generally good correspondence (higher correlation in the log2 ratios) of one replicate versus another in the MessageAmp II arrays relative to the Affymetrix arrays.

Figure 6. Scatter Plot of log2 Ratios of HeLa vs. Brain for MessageAmp II and Affymetrix Standard Protocol. Expression values were estimated using RMA. Log2 (HeLa/Brain) are plotted for each protocol represent the comparisons possible where the ratios are derived
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