Rapid Development of Real-Time RT-PCR Assays Using Universal ProbeLibrary: Applications for Dissecting Signaling Pathways by RNA interference

摘要

To characterize gene function on a genome-wide scale, reliable and rapid methods to quantify gene expression levels are required. A key advance in functional genomics has been the use of RNA interference (RNAi), which allows the silencing of genes in metazoans through the introduction of short, double-stranded (ds) RNAs homologous to endogenous target mRNAs [1]. RNAi has been successfully used to silence gene expression in invertebrate and mammalian cultured cells, thereby enabling loss-of-function studies that were previously not feasible. RNAi has become a widely used approach to study the phenotypic effects of knocked down components of many pathways implicated in physiology and disease. Stringent quality-control procedures to monitor RNAi experiments are essential. Requirements for such a methodology are Ca) high flexibility in assay design if many different genes need to be examined, (b] precision in assessing gene expression levels, and (cj ability to measure expression levels of multiple genes in parallel.