High-Throughput Profiling of Mature MicroRNA by Real-Time PCR

Abstract

Real-time quantitative PCR has become a staple technique of most molecular biology laboratories. Configuration of quantitative PCR instruments into 384-well plates has allowed the technology to function as a low-density gene expression array. In this chapter, we present protocols and data that apply quantitative PCR to profile hundreds of genes simultaneously. TaqMan probe and primer sets were pipetted individually into 384-well reaction plates using liquid-handling robots. This substantially increased throughput and reduced error. This protocol was used to expression profile mature miRNAs in total RNA isolated from circulating microvesicles and in peripheral blood mononuclear cells (PBMCs) of healthy donors. Using a robotics system to load the 384-well plates into the quantitative PCR instrument, 420 miRNAs were profiled in RNA isolated from microvesicles and PBMCs of 50 patients in about 2 weeks. Using equipment located in many gene expression laboratories or core facilities, low-density gene expression profiling may be easily achieved with minimal error.