Analysis of Affymetrix GeneChip® Data Using Amplified RNA

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Abstract
When small biological samples are collected by microdissection or other methods, amplification techniques are required to provide sufficient target for hybridization to expression arrays. One such technique is to perform two successive rounds of T7-based in vitro transcription. However, the use of random primers, required to regenerate cDNA from the first round of transcription, results in shortened copies of cDNA from which the 5′ end is missing. In this paper we describe an experiment designed to compare the quality of data obtained from labeling small RNA samples using the Affymetrix Two-Cycle Eukaryotic Target Labeling procedure to that of data obtained using the One-Cycle Eukaryotic Target Labeling protocol. We utilized different preprocessing algorithms to compare the data generated using both labeling methods and present a new algorithm that improves upon existing ones in this setting. Pre-Microarray RNA Amplification Regular readers of BioTechniques will be well aware of the ongoing advances in the reliable amplification of RNA for use in microarray experiments. These procedures allow submicrogram amounts of RNA (such as that obtained when small biological samples are collected by microdissection) to be faithfully amplified to levels appropriate for microarray analysis. Although there has been substantial progress in development and validation of these wet-lab techniques, fewer studies have examined optimal data processing procedures for such situations. To address this gap, Cope et al. (p. 165) examined the performance of four different processing algorithms on RNA prepared for Affymetrix GeneChips® using either the standard method or a two-cycle amplification procedure. The findings are reassuring, in that by every measure tested, the two-cycle amplification protocol does not significantly lessen the quality of the hybridization. In addition, analytical methods that work well for the...