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Is RNA Amplification Necessary for Microarrays?

13,284 human cDNA elements.

Analysis of differential gene expression in the cells treated with TNF-α compared to untreated control cells identified 1296 genes in the amplified samples and 155 genes in the unamplified samples whose expression was affected by cytokine treatment. By comparing genes identified with probe prepared by both amplified and unamplified RNA, three categories were created: "genes common in amplified and unamplified RNA", "genes unique to amplified RNA", and "genes unique to unamplified RNA" (Figure 1). Real-time quantitative RT-PCR (qRT-PCR) analysis using SYBR Green detection of expression differences of a subset of the genes from each category was carried out. A summary of the observed results from qRT-PCR analysis and array analysis is shown in Table 1. The following conclusions were drawn from these validation studies:

(A) Because genes were identified as having differential expression with both preparations of labeled target, "common identified genes" are considered to have the highest probability of being verifiably regulated by TNF-α. In general, the ratio of gene expression with and without TNF-α treatment from amplified RNA had a closer correlation with the qRT-PCR data than ratios from unamplified RNA. 94% of the "common identified genes" show similar kinase-regulation patterns between amplified and unamplified samples.

(B) "Genes unique to
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