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Direct Cell Lysis for Single-Cell Gene Expression Profiling

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...

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Autores principales: Svec, David, Andersson, Daniel, Pekny, Milos, Sjöback, Robert, Kubista, Mikael, Ståhlberg, Anders
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3819639/
https://www.ncbi.nlm.nih.gov/pubmed/24224157
http://dx.doi.org/10.3389/fonc.2013.00274
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author Svec, David
Andersson, Daniel
Pekny, Milos
Sjöback, Robert
Kubista, Mikael
Ståhlberg, Anders
author_facet Svec, David
Andersson, Daniel
Pekny, Milos
Sjöback, Robert
Kubista, Mikael
Ståhlberg, Anders
author_sort Svec, David
collection PubMed
description 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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spelling pubmed-38196392013-11-09 Direct Cell Lysis for Single-Cell Gene Expression Profiling Svec, David Andersson, Daniel Pekny, Milos Sjöback, Robert Kubista, Mikael Ståhlberg, Anders Front Oncol Oncology 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. Frontiers Media S.A. 2013-11-07 /pmc/articles/PMC3819639/ /pubmed/24224157 http://dx.doi.org/10.3389/fonc.2013.00274 Text en Copyright © 2013 Svec, Andersson, Pekny, Sjöback, Kubista and Ståhlberg. http://creativecommons.org/licenses/by/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Oncology
Svec, David
Andersson, Daniel
Pekny, Milos
Sjöback, Robert
Kubista, Mikael
Ståhlberg, Anders
Direct Cell Lysis for Single-Cell Gene Expression Profiling
title Direct Cell Lysis for Single-Cell Gene Expression Profiling
title_full Direct Cell Lysis for Single-Cell Gene Expression Profiling
title_fullStr Direct Cell Lysis for Single-Cell Gene Expression Profiling
title_full_unstemmed Direct Cell Lysis for Single-Cell Gene Expression Profiling
title_short Direct Cell Lysis for Single-Cell Gene Expression Profiling
title_sort direct cell lysis for single-cell gene expression profiling
topic Oncology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3819639/
https://www.ncbi.nlm.nih.gov/pubmed/24224157
http://dx.doi.org/10.3389/fonc.2013.00274
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