Advanced

High salt buffer improves integrity of RNA after fluorescence-activated cell sorting of intracellular labeled cells.

Nilsson, Helén LU ; Krawczyk, Krzysztof LU and Johansson, Martin E (2014) In Journal of Biotechnology 192(Sep 30). p.62-65
Abstract
Over the past years, massive progress has been made in the ability to collect large-scale gene expression data from a limited sample size. Combined with improvements in multiplex flow cytometry-based techniques, this has made it possible to isolate and characterize specific cellular subtypes within heterogeneous populations, with a great impact on our understanding of different biological processes. However, sorting based on intracellular markers requires fixation and permeabilization of samples, and very often the integrity of RNA molecules is compromised during this process. Many attempts have been made to improve the quality of nucleic acids from such samples, but RNA degradation still remains a limiting factor for downstream analyses.... (More)
Over the past years, massive progress has been made in the ability to collect large-scale gene expression data from a limited sample size. Combined with improvements in multiplex flow cytometry-based techniques, this has made it possible to isolate and characterize specific cellular subtypes within heterogeneous populations, with a great impact on our understanding of different biological processes. However, sorting based on intracellular markers requires fixation and permeabilization of samples, and very often the integrity of RNA molecules is compromised during this process. Many attempts have been made to improve the quality of nucleic acids from such samples, but RNA degradation still remains a limiting factor for downstream analyses. Here we present a method to isolate high quality RNA from cells that have been fixed, permeabilized, intracellularly labeled and sorted. By performing all incubation steps in the presence of a high salt buffer, RNA degradation was avoided and samples with remarkable integrity were obtained. This procedure offers a straightforward and very affordable technique to retrieve high quality RNA from isolated cell populations, which increases the possibilities to characterize gene expression profiles of subpopulations from mixed samples, a technique with implications in a broad range of research fields. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Biotechnology
volume
192
issue
Sep 30
pages
62 - 65
publisher
Elsevier
external identifiers
  • pmid:25277986
  • wos:000345970300011
  • scopus:84909635513
ISSN
1873-4863
DOI
10.1016/j.jbiotec.2014.09.016
language
English
LU publication?
yes
id
a3c5cd62-a617-4295-a50c-5ab276b4b694 (old id 4738497)
alternative location
http://www.ncbi.nlm.nih.gov/pubmed/25277986?dopt=Abstract
date added to LUP
2014-11-05 18:36:36
date last changed
2017-07-23 03:05:09
@article{a3c5cd62-a617-4295-a50c-5ab276b4b694,
  abstract     = {Over the past years, massive progress has been made in the ability to collect large-scale gene expression data from a limited sample size. Combined with improvements in multiplex flow cytometry-based techniques, this has made it possible to isolate and characterize specific cellular subtypes within heterogeneous populations, with a great impact on our understanding of different biological processes. However, sorting based on intracellular markers requires fixation and permeabilization of samples, and very often the integrity of RNA molecules is compromised during this process. Many attempts have been made to improve the quality of nucleic acids from such samples, but RNA degradation still remains a limiting factor for downstream analyses. Here we present a method to isolate high quality RNA from cells that have been fixed, permeabilized, intracellularly labeled and sorted. By performing all incubation steps in the presence of a high salt buffer, RNA degradation was avoided and samples with remarkable integrity were obtained. This procedure offers a straightforward and very affordable technique to retrieve high quality RNA from isolated cell populations, which increases the possibilities to characterize gene expression profiles of subpopulations from mixed samples, a technique with implications in a broad range of research fields.},
  author       = {Nilsson, Helén and Krawczyk, Krzysztof and Johansson, Martin E},
  issn         = {1873-4863},
  language     = {eng},
  number       = {Sep 30},
  pages        = {62--65},
  publisher    = {Elsevier},
  series       = {Journal of Biotechnology},
  title        = {High salt buffer improves integrity of RNA after fluorescence-activated cell sorting of intracellular labeled cells.},
  url          = {http://dx.doi.org/10.1016/j.jbiotec.2014.09.016},
  volume       = {192},
  year         = {2014},
}