Single-cell gene expression profiling using reverse transcription quantitative real-time PCR.
Even in an apparently homogeneous population of cells there are considerable differences between individual cells. A response to a stimulus of a cell population or tissue may be consistent and gradual while the single-cell response might be binary and apparently irregular. The origin of this variability may be preprogrammed or stochastic and a study of this phenomenon will require quantitative measurements of individual cells. Here, we describe a method to collect dispersed single cells either by glass capillaries or flow cytometry, followed by quantitative mRNA profiling using reverse transcription and real-time PCR. We present a single cell lysis protocol and optimized priming conditions for reverse transcription. The large cell-to-cell variability in single-cell gene expression measurements excludes it from standard data analysis. Correlation studies can be used to find common regulatory elements that are indistinguishable at the population level. Single-cell gene expression profiling has the potential to become common practice in many laboratories and a powerful research tool for deeper understanding of molecular mechanisms.
Ståhlberg
Anders
A
Lundberg Laboratory for Cancer, Department of Pathology, Sahlgrenska Academy at University of Gothenburg, Gula Straket 8, 413 45 Gothenburg, Sweden. anders.stahlberg@neuro.gu.se
Bengtsson
Martin
M
D002469:Cell Separation; D005434:Flow Cytometry; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D048348:Reverse Transcription
D012333:RNA, Messenger