Improved quantitative real-time RT-PCR for expression profiling 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 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.
University Laboratory of Physiology and MRC Anatomical Neuropharmacology Unit, Department of Pharmacology, Oxford University, Parks Road, Oxford OX1 3PT, UK. birgit.liss@physiol.ox.ac.uk
Birgit
B
Liss
D000818:Animals; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D018076:DNA, Complementary; D012333:RNA, Messenger