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Comparison of procedures for RNA-extraction from peripheral blood mononuclear cells

RNA quality and quantity are important factors for ensuring the accuracy of gene expression analysis and other RNA-based downstream applications. Thus far, only a limited number of methodological studies have compared sample storage and RNA extraction procedures for human cells. We compared three co...

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Autores principales: Rodríguez, Antonio, Duyvejonck, Hans, Van Belleghem, Jonas D., Gryp, Tessa, Van Simaey, Leen, Vermeulen, Stefan, Van Mechelen, Els, Vaneechoutte, Mario
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7034890/
https://www.ncbi.nlm.nih.gov/pubmed/32084228
http://dx.doi.org/10.1371/journal.pone.0229423
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author Rodríguez, Antonio
Duyvejonck, Hans
Van Belleghem, Jonas D.
Gryp, Tessa
Van Simaey, Leen
Vermeulen, Stefan
Van Mechelen, Els
Vaneechoutte, Mario
author_facet Rodríguez, Antonio
Duyvejonck, Hans
Van Belleghem, Jonas D.
Gryp, Tessa
Van Simaey, Leen
Vermeulen, Stefan
Van Mechelen, Els
Vaneechoutte, Mario
author_sort Rodríguez, Antonio
collection PubMed
description RNA quality and quantity are important factors for ensuring the accuracy of gene expression analysis and other RNA-based downstream applications. Thus far, only a limited number of methodological studies have compared sample storage and RNA extraction procedures for human cells. We compared three commercially available RNA extraction kits, i.e., (NucliSENS) easyMAG, RNeasy (Mini Kit) and RiboPure (RNA Purification Kit–blood). In addition, additional conditions, such as storage medium and storage temperature of human peripheral blood mononuclear cells were evaluated, i.e., 4 °C for RNAlater or -80 °C for QIAzol and for the respective cognate lysis buffers; easyMAG, RNeasy or RiboPure. RNA was extracted from aliquots that had been stored for one day (Run 1) or 83 days (Run 2). After DNase treatment, quantity and quality of RNA were assessed by means of a NanoDrop spectrophotometer, 2100 Bioanalyzer and RT-qPCR for the ACTB reference gene. We observed that high-quality RNA can be obtained using RNeasy and RiboPure, regardless of the storage medium, whereas samples stored in RNAlater resulted in the least amount of RNA extracted. In addition, RiboPure combined with storage of samples in its cognate lysis buffer yielded twice as much RNA as all other procedures. These results were supported by RT-qPCR and by the reproducibility observed for two independent extraction runs.
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spelling pubmed-70348902020-02-27 Comparison of procedures for RNA-extraction from peripheral blood mononuclear cells Rodríguez, Antonio Duyvejonck, Hans Van Belleghem, Jonas D. Gryp, Tessa Van Simaey, Leen Vermeulen, Stefan Van Mechelen, Els Vaneechoutte, Mario PLoS One Research Article RNA quality and quantity are important factors for ensuring the accuracy of gene expression analysis and other RNA-based downstream applications. Thus far, only a limited number of methodological studies have compared sample storage and RNA extraction procedures for human cells. We compared three commercially available RNA extraction kits, i.e., (NucliSENS) easyMAG, RNeasy (Mini Kit) and RiboPure (RNA Purification Kit–blood). In addition, additional conditions, such as storage medium and storage temperature of human peripheral blood mononuclear cells were evaluated, i.e., 4 °C for RNAlater or -80 °C for QIAzol and for the respective cognate lysis buffers; easyMAG, RNeasy or RiboPure. RNA was extracted from aliquots that had been stored for one day (Run 1) or 83 days (Run 2). After DNase treatment, quantity and quality of RNA were assessed by means of a NanoDrop spectrophotometer, 2100 Bioanalyzer and RT-qPCR for the ACTB reference gene. We observed that high-quality RNA can be obtained using RNeasy and RiboPure, regardless of the storage medium, whereas samples stored in RNAlater resulted in the least amount of RNA extracted. In addition, RiboPure combined with storage of samples in its cognate lysis buffer yielded twice as much RNA as all other procedures. These results were supported by RT-qPCR and by the reproducibility observed for two independent extraction runs. Public Library of Science 2020-02-21 /pmc/articles/PMC7034890/ /pubmed/32084228 http://dx.doi.org/10.1371/journal.pone.0229423 Text en https://creativecommons.org/publicdomain/zero/1.0/ This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 (https://creativecommons.org/publicdomain/zero/1.0/) public domain dedication.
spellingShingle Research Article
Rodríguez, Antonio
Duyvejonck, Hans
Van Belleghem, Jonas D.
Gryp, Tessa
Van Simaey, Leen
Vermeulen, Stefan
Van Mechelen, Els
Vaneechoutte, Mario
Comparison of procedures for RNA-extraction from peripheral blood mononuclear cells
title Comparison of procedures for RNA-extraction from peripheral blood mononuclear cells
title_full Comparison of procedures for RNA-extraction from peripheral blood mononuclear cells
title_fullStr Comparison of procedures for RNA-extraction from peripheral blood mononuclear cells
title_full_unstemmed Comparison of procedures for RNA-extraction from peripheral blood mononuclear cells
title_short Comparison of procedures for RNA-extraction from peripheral blood mononuclear cells
title_sort comparison of procedures for rna-extraction from peripheral blood mononuclear cells
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7034890/
https://www.ncbi.nlm.nih.gov/pubmed/32084228
http://dx.doi.org/10.1371/journal.pone.0229423
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