An Integrated High-Throughput System for mRNA Purification and Quantitation for Use in Identifying Gene Knockdown by RNA Interference

Abstract

Expression profiling and RNA interference (RNAi) studies require rapid and accurate ways to profile changes in gene expression. Such studies use methodologies for the isolation and subsequent analysis of mRNA because it is central to the transfer of genetic information within a cell. Quantitative reverse transcriptional PCR (RT-PCR) is routinely used to verify knockdown of gene expression mediated by RNAi, but its success is dependent upon the quality of the purified mRNA template. An automated system has been developed for the isolation and subsequent analysis of the mRNA. This system uses Sigma's SpyLine Poly A+ Capture Kit, a novel system for the rapid isolation of poly A+ mRNA from cultured mammalian cells for direct use in quantitative RT-PCR analysis. The automated method was used to identify effective RNAi gene knockdown. Results indicate that this approach has both the sensitivity and reproducibility necessary for measuring transcript levels following gene knockdown.