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Direct cell lysis for single-cell gene expression profiling.

Journal article
Authors David Svec
Daniel Andersson
Milos Pekny
Robert Sjöback
Mikael Kubista
Anders Ståhlberg
Published in Frontiers in oncology
Volume 3
Pages 274
ISSN 2234-943X
Publication year 2013
Published at Institute of Neuroscience and Physiology, Department of Clinical Neuroscience and Rehabilitation
Sahlgrenska Cancer Center
Pages 274
Language en
Links dx.doi.org/10.3389/fonc.2013.00274
Keywords real-time PCR, single-cell biology, single-cell gene expression, RNA spike, DNA spike, cell lysis, direct lysis, RNA purification
Subject categories Cell and Molecular Biology, Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

Abstract

The interest to analyze single and few cell samples is rapidly increasing. Numerous extraction protocols to purify nucleic acids are available, but most of them compromise severely on yield to remove contaminants and are therefore not suitable for the analysis of samples containing small numbers of transcripts only. Here, we evaluate 17 direct cell lysis protocols for transcript yield and compatibility with downstream reverse transcription quantitative real-time PCR. Four endogenously expressed genes are assayed together with RNA and DNA spikes in the samples. We found bovine serum albumin (BSA) to be the best lysis agent, resulting in efficient cell lysis, high RNA stability, and enhanced reverse transcription efficiency. Furthermore, we found direct cell lysis with BSA superior to standard column based extraction methods, when analyzing from 1 up to 512 mammalian cells. In conclusion, direct cell lysis protocols based on BSA can be applied with most cell collection methods and are compatible with most analytical workflows to analyze single-cells as well as samples composed of small numbers of cells.

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