Methodology article

Evaluation of sense-strand mRNA amplification by comparative quantitative PCR

Loyal A Goff¹ , Jessica Bowers² , Jaime Schwalm² , Kevin Howerton² , Robert C Getts² and Ronald P Hart¹

¹W.M. Keck Center for Collaborative Neuroscience, Rutgers University, Piscataway, NJ 08854 USA

²Genisphere, Inc., Hatfield, PA 19440 USA

author email corresponding author email

BMC Genomics 2004, 5:76doi:10.1186/1471-2164-5-76

Published:	6 October 2004

Abstract

Background

RNA amplification is required for incorporating laser-capture microdissection techniques into microarray assays. However, standard oligonucleotide microarrays contain sense-strand probes, so traditional T7 amplification schemes producing anti-sense RNA are not appropriate for hybridization when combined with conventional reverse transcription labeling methods. We wished to assess the accuracy of a new sense-strand RNA amplification method by comparing ratios between two samples using quantitative real-time PCR (qPCR), mimicking a two-color microarray assay.

Results

We performed our validation using qPCR. Three samples of rat brain RNA and three samples of rat liver RNA were amplified using several kits (Ambion messageAmp, NuGen Ovation, and several versions of Genisphere SenseAmp). Results were assessed by comparing the liver/brain ratio for 192 mRNAs before and after amplification. In general, all kits produced strong correlations with unamplified RNAs. The SenseAmp kit produced the highest correlation, and was also able to amplify a partially degraded sample accurately.

Conclusion

We have validated an optimized sense-strand RNA amplification method for use in comparative studies such as two-color microarrays.