Experimental design for testing RNA amplification techniques. The scheme for estimating errors and testing the performance of each amplification technique in generating microarray expression data is shown. Total RNA was isolated from two mouse cell lines, an ovary epithelial cell line (OV) and 3T3 fibroblasts (3T3). Unamplified targets were synthesized from 100 μg of total RNA by reverse transcription. RNA was amplified by using linear T7 based- or exponential PCR-based amplification (see text for details). Synthesized DNA/cRNA samples were indirectly labeled with Cy3/Cy5 fluors. Labeled targets were co-hybridized on oligonucleotide arrays (see Materials and methods for details). Design 1, same vs same control hybridization. Labeled DNA/cRNA were divided in two parts and each was coupled with either Cy3 or Cy5 NHS-esters followed by co-hybridization on the same slide. Design 2, hybridization of technical replicates. Pairs of technical replicates synthesized from ovarian cell line total RNA by each amplification method or by reverse transcription (unamplified cDNA) were compared. Design 3, comparison of gene expression between two different cell lines by hybridization of cDNAs amplified by the same technique.
Subkhankulova and Livesey Genome Biology 2006 7:R18 doi:10.1186/gb-2006-7-3-r18