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Is RNA Amplification Necessary for Microarrays?

ether the individual genes that were differentially expressed in amplified samples would have been identified as such using total RNA. For this experiment, the number of "outliers" defined by authors as "A gene with expression ratios of greater than or equal to 2.0 or less than or equal to 0.5 in two reciprocal microarray analysis" identified using total RNA or amplified RNA (one or two rounds of amplification) were compared. The amplified samples identified approximately 80% of the genes selected as outliers with total RNA, with an 80% concordance between target made by one and two rounds of amplification. Thus amplification yielded approximately 2 times more outliers than total RNA, with 80% of these showing similar trends in expression between amplified and total RNA samples. Validation of the "outliers" between total RNA and amplified RNA was carried out using real-time PCR analysis. The PCR data suggested that the majority of outliers identified using amplified RNA are valid. Thus Feldman et al. also conclude that all RNA samples should undergo at least one round of amplification to maximize the quality of array data for detection of differentially expressed genes.

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