Improved quantitative real-time RT-PCR for expression profiling of individual cells

Abstract

The real‐time quantitative polymerase chain reaction (rtqPCR) has overcome the limitations of conventional, time‐consuming quantitative PCR strategies and is maturing into a routine tool to quantify gene expression levels, following reverse transcription (RT) of mRNA into complementary DNA (cDNA). Expression profiling with single‐cell resolution is highly desirable, in particular for complex tissues like the brain that contain a large variety of different cell types in close proximity. The patch‐clamp technique allows selective harvesting of single‐cell cytoplasm after recording of cellular activity. However, components of the cDNA reaction, in particular the reverse transcriptase itself, significantly inhibit subsequent rtqPCR amplification. Using undiluted single‐cell cDNA reaction mix directly as template for rtqPCR, I observed that the amplification kinetics of rtqPCRs were dramatically altered in a non‐systematic fashion. Here, I describe a simple and robust precipitation protocol suitable for purification of single‐cell cDNA that completely removes inhibitory RT components without detectable loss of cDNA. This improved single‐cell real‐time RT–PCR protocol provides a powerful tool to quantify differential gene expression of individual cells and thus could complement global microarray‐based expression profiling strategies.