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Quantitation of Gene Expression in Formaldehyde-Fixed and Fluorescence-Activated Sorted Cells

Fluorescence-activated cell sorting (FACS) is a sensitive and valuable technique to characterize cellular subpopulations and great advances have been made using this approach. Cells are often fixed with formaldehyde prior to the sorting process to preserve cell morphology and maintain the expression...

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Detalles Bibliográficos
Autores principales: Russell, Julia N., Clements, Janice E., Gama, Lucio
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3759445/
https://www.ncbi.nlm.nih.gov/pubmed/24023909
http://dx.doi.org/10.1371/journal.pone.0073849
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author Russell, Julia N.
Clements, Janice E.
Gama, Lucio
author_facet Russell, Julia N.
Clements, Janice E.
Gama, Lucio
author_sort Russell, Julia N.
collection PubMed
description Fluorescence-activated cell sorting (FACS) is a sensitive and valuable technique to characterize cellular subpopulations and great advances have been made using this approach. Cells are often fixed with formaldehyde prior to the sorting process to preserve cell morphology and maintain the expression of surface molecules, as well as to ensure safety in the sorting of infected cells. It is widely recognized that formaldehyde fixation alters RNA and DNA structure and integrity, thus analyzing gene expression in these cells has been difficult. We therefore examined the effects of formaldehyde fixation on the stability and quantitation of nucleic acids in cell lines, primary leukocytes and also cells isolated from SIV-infected pigtailed macaques. We developed a method to extract RNA from fixed cells that yielded the same amount of RNA as our common method of RNA isolation from fresh cells. Quantitation of RNA by RT-qPCR in fixed cells was not always comparable with that in unfixed cells. In comparison, when RNA was measured by the probe-based NanoString system, there was no significant difference in RNA quantitation. In addition, we demonstrated that quantitation of proviral DNA in fixed cells by qPCR is comparable to that in unfixed cells when normalized by a single-copy cellular gene. These results provide a systematic procedure to quantitate gene expression in cells that have been fixed with formaldehyde and sorted by FACS.
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spelling pubmed-37594452013-09-10 Quantitation of Gene Expression in Formaldehyde-Fixed and Fluorescence-Activated Sorted Cells Russell, Julia N. Clements, Janice E. Gama, Lucio PLoS One Research Article Fluorescence-activated cell sorting (FACS) is a sensitive and valuable technique to characterize cellular subpopulations and great advances have been made using this approach. Cells are often fixed with formaldehyde prior to the sorting process to preserve cell morphology and maintain the expression of surface molecules, as well as to ensure safety in the sorting of infected cells. It is widely recognized that formaldehyde fixation alters RNA and DNA structure and integrity, thus analyzing gene expression in these cells has been difficult. We therefore examined the effects of formaldehyde fixation on the stability and quantitation of nucleic acids in cell lines, primary leukocytes and also cells isolated from SIV-infected pigtailed macaques. We developed a method to extract RNA from fixed cells that yielded the same amount of RNA as our common method of RNA isolation from fresh cells. Quantitation of RNA by RT-qPCR in fixed cells was not always comparable with that in unfixed cells. In comparison, when RNA was measured by the probe-based NanoString system, there was no significant difference in RNA quantitation. In addition, we demonstrated that quantitation of proviral DNA in fixed cells by qPCR is comparable to that in unfixed cells when normalized by a single-copy cellular gene. These results provide a systematic procedure to quantitate gene expression in cells that have been fixed with formaldehyde and sorted by FACS. Public Library of Science 2013-09-02 /pmc/articles/PMC3759445/ /pubmed/24023909 http://dx.doi.org/10.1371/journal.pone.0073849 Text en © 2013 Russell et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Russell, Julia N.
Clements, Janice E.
Gama, Lucio
Quantitation of Gene Expression in Formaldehyde-Fixed and Fluorescence-Activated Sorted Cells
title Quantitation of Gene Expression in Formaldehyde-Fixed and Fluorescence-Activated Sorted Cells
title_full Quantitation of Gene Expression in Formaldehyde-Fixed and Fluorescence-Activated Sorted Cells
title_fullStr Quantitation of Gene Expression in Formaldehyde-Fixed and Fluorescence-Activated Sorted Cells
title_full_unstemmed Quantitation of Gene Expression in Formaldehyde-Fixed and Fluorescence-Activated Sorted Cells
title_short Quantitation of Gene Expression in Formaldehyde-Fixed and Fluorescence-Activated Sorted Cells
title_sort quantitation of gene expression in formaldehyde-fixed and fluorescence-activated sorted cells
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3759445/
https://www.ncbi.nlm.nih.gov/pubmed/24023909
http://dx.doi.org/10.1371/journal.pone.0073849
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