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Improving RNA Amplification

otocols was equivalent to that seen within Affymetrix replicates.

Figure 7. Comparison Between and Within Protocols of Mean Percent Shared Present and Absent Calls. 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. The right most column contains the total concordance, which is the number of concordant calls (P-P, A-A, or M-M) divided by the total number of calls (or genes = 8793). (Present/Absent calls were determined with Affymetrix GeneChip operating software (GCOS) for all arrays.)


IVT incubation time vs. Microarray Signals
While a shortened IVT time may produce sufficient cRNA for microarray hybridization, the signal characteristics of the labeled cRNA on a microarray is the critical factor for determining functionality. A series of three IVT time points (4, 6, and 14 hr) using total RNA from brain and HeLa were tested to measure whether any difference was observed between IVT times. Total RNA input amounts used were 100, 200 and 1000 ng. These samples were amplified and labeled as above, then hybridized to GeneChip Human Genome Focus Arrays. Again, for a reference series we used the Affymetrix protocol with duplicate 1000 ng reactions of the same RNA. Fourteen arrays were scanned and signal intensities were analyzed for similarity.


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