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Comparison of methods for miRNA isolation and quantification from ovine plasma

microRNA (miRNA) are promising candidates for disease biomarkers as they are abundant in circulation, highly stable in biological fluids and may yield diagnostic biomarker signatures. The reported issues with miRNA isolation using traditional RNA reagents necessitates the optimisation of miRNA isola...

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Autores principales: Wright, Kathryn, de Silva, Kumudika, Purdie, Auriol C., Plain, Karren M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6972740/
https://www.ncbi.nlm.nih.gov/pubmed/31964966
http://dx.doi.org/10.1038/s41598-020-57659-7
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author Wright, Kathryn
de Silva, Kumudika
Purdie, Auriol C.
Plain, Karren M.
author_facet Wright, Kathryn
de Silva, Kumudika
Purdie, Auriol C.
Plain, Karren M.
author_sort Wright, Kathryn
collection PubMed
description microRNA (miRNA) are promising candidates for disease biomarkers as they are abundant in circulation, highly stable in biological fluids and may yield diagnostic biomarker signatures. The reported issues with miRNA isolation using traditional RNA reagents necessitates the optimisation of miRNA isolation from challenging samples. In this study we compared six commercial RNA extraction kits to evaluate their ability to isolate miRNA from ovine plasma. We also compared three methods for quantification of small RNA extracted from plasma to determine the most reliable. Using minimal sample inputs of fresh and frozen plasma from five sheep, we compared the six kits (Kit A-F) using quantitative PCR. Operational factors were also assessed for each kit. Kits A and B provided the best detection of the miRNA qPCR reference genes across fresh and frozen samples (p < 0.001) followed by Kit C. The Qubit and microRNA assay provided the least variation (% CV 5.47, SEM ± 0.07), followed by the NanoDrop (% CV 7.01, SEM ± 0.92) and Agilent Bioanalyzer (% CV 59.21, SEM ± 1.31). We identify Kit A to be optimal for isolating miRNA from small volumes of fresh and frozen ovine plasma, and Kit B the top performing kit taking into consideration miRNA detection and operational factors. The Qubit fluorometer using a microRNA assay was the most reliable miRNA quantification method.
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spelling pubmed-69727402020-01-27 Comparison of methods for miRNA isolation and quantification from ovine plasma Wright, Kathryn de Silva, Kumudika Purdie, Auriol C. Plain, Karren M. Sci Rep Article microRNA (miRNA) are promising candidates for disease biomarkers as they are abundant in circulation, highly stable in biological fluids and may yield diagnostic biomarker signatures. The reported issues with miRNA isolation using traditional RNA reagents necessitates the optimisation of miRNA isolation from challenging samples. In this study we compared six commercial RNA extraction kits to evaluate their ability to isolate miRNA from ovine plasma. We also compared three methods for quantification of small RNA extracted from plasma to determine the most reliable. Using minimal sample inputs of fresh and frozen plasma from five sheep, we compared the six kits (Kit A-F) using quantitative PCR. Operational factors were also assessed for each kit. Kits A and B provided the best detection of the miRNA qPCR reference genes across fresh and frozen samples (p < 0.001) followed by Kit C. The Qubit and microRNA assay provided the least variation (% CV 5.47, SEM ± 0.07), followed by the NanoDrop (% CV 7.01, SEM ± 0.92) and Agilent Bioanalyzer (% CV 59.21, SEM ± 1.31). We identify Kit A to be optimal for isolating miRNA from small volumes of fresh and frozen ovine plasma, and Kit B the top performing kit taking into consideration miRNA detection and operational factors. The Qubit fluorometer using a microRNA assay was the most reliable miRNA quantification method. Nature Publishing Group UK 2020-01-21 /pmc/articles/PMC6972740/ /pubmed/31964966 http://dx.doi.org/10.1038/s41598-020-57659-7 Text en © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Wright, Kathryn
de Silva, Kumudika
Purdie, Auriol C.
Plain, Karren M.
Comparison of methods for miRNA isolation and quantification from ovine plasma
title Comparison of methods for miRNA isolation and quantification from ovine plasma
title_full Comparison of methods for miRNA isolation and quantification from ovine plasma
title_fullStr Comparison of methods for miRNA isolation and quantification from ovine plasma
title_full_unstemmed Comparison of methods for miRNA isolation and quantification from ovine plasma
title_short Comparison of methods for miRNA isolation and quantification from ovine plasma
title_sort comparison of methods for mirna isolation and quantification from ovine plasma
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6972740/
https://www.ncbi.nlm.nih.gov/pubmed/31964966
http://dx.doi.org/10.1038/s41598-020-57659-7
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