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Improving RNA Amplification

p II protocol. First, the relative consistency of the MessageAmp II protocol was measured by comparing replicate arrays, where amplified samples originated from the same RNA source, either inferior temporal cortex from human brain (brain) or HeLa S3 cells (HeLa). These cRNA samples were hybridized to GeneChip Human Genome Focus Arrays following the Affymetrix guidelines. For a reference series we used the Affymetrix GeneChip 3'-Amplification Reagents One-Cycle cDNA Synthesis Kit and IVT Labeling Reagents (from here on referred to as the Affymetrix protocol) with duplicate 1000 ng reactions of the same RNA. The Affymetrix quality assessment guidelines were met for all arrays in this study. This included 3'/5' ratios (under 2.0), percent Present calls (50-53%), scaling factors, average background, as well as visual inspection of images. This assessment indicated that all arrays were of high quality.

MessageAmp II Reproducibility. Signal values for each probe set were converted from .CEL files into a summary of expression values measured for each of the 8793 probe sets (genes). Expression values were estimated using Robust Multi-array Average (RMA) [4-6] as implemented in BioConductor (www.bioconductor.org). The correlation established by this comparison shows that amplification using these protocols with 1000 ng of input total RNA is highly reproducible (Figure 5).

Figure 5. (A) Replicate Analysis of Two Amplification Protocols. Normalized signals are plotted from replicate amplificatio
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