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Single-cell gene expression profiling using reverse transcription quantitative real-time PCR.

Review article
Authors Anders Ståhlberg
Martin Bengtsson
Published in Methods (San Diego, Calif.)
Volume 50
Issue 4
Pages 282-8
ISSN 1095-9130
Publication year 2010
Published at Institute of Biomedicine, Department of Pathology
Pages 282-8
Language en
Links dx.doi.org/10.1016/j.ymeth.2010.01....
Keywords Cell Separation, Flow Cytometry, Gene Expression Profiling, methods, Gene Expression Regulation, RNA, Messenger, genetics, Reverse Transcriptase Polymerase Chain Reaction, methods, Reverse Transcription
Subject categories Cell and Molecular Biology

Abstract

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.

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