Improved quantitative real-time RT±PCR for expression pro®ling of individual cells

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 pro®ling 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, signi®cantly inhibit subsequent rtqPCR ampli®cation. Using undiluted single-cell cDNA reaction mix directly as template for rtqPCR, I observed that the ampli®cation kinetics of rtqPCRs were dramatically altered in a non-systematic fashion. Here, I describe a simple and robust precipitation protocol suitable for puri®cation 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 pro®ling strategies.