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Improving RNA Amplification

from unique arrays. HeLa and brain RNAs were amplified as stated in the MessageAmp II and Affymetrix protocols.


These two analyses (Figures 5 and 6) indicate that the two protocols are reproducible and comparisons made between the protocols do not appear to have any systematic differences above the observed variation between replicates within a protocol.

Present Absent Call Concordance. We used two comparisons to evaluate Present/Absent call concordance: 1) the correspondence of Present (P) and Absent (A) calls comparing the MessageAmp II and Affymetrix standard protocols to address consistency between protocols, and 2) the correspondence within the MessageAmp II and Affymetrix protocols. The P-A calls are based on the Wilcoxon signed rank-based gene expression Present/Absent detection algorithm first implemented in the Affymetrix Microarray Suite, Version 5. This was done separately by RNA type (brain and HeLa). We paired arrays in all possible combinations and summarized the average performance. When comparing two arrays, the percent shared Present (PSP) calls is the total genes called present in both arrays divided by the total number of (unique) genes called present between arrays. An equivalent statistic was used for the percent shared Absent (PSA) calls; for this analysis we ignored Marginal (M) calls. Also included was total concordance, calculated using all call types by simply dividing the number of concordant calls (P-P, A-A, or M-M) by the total number of calls (or genes = 8793). The results, shown in Figure 7, demonstrate again that the correspondence between the MessageAmp II and Affymetrix pr
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