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Quantification of microRNA levels in plasma – Impact of preanalytical and analytical conditions
Numerous studies have reported a potential role for circulating microRNAs as biomarkers in a wide variety of diseases. However, there is a critical reproducibility challenge some of which might be due to differences in preanalytical and/or analytical factors. Thus, in the current study we systematic...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6053236/ https://www.ncbi.nlm.nih.gov/pubmed/30024941 http://dx.doi.org/10.1371/journal.pone.0201069 |
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author | Binderup, Helle Glud Madsen, Jonna Skov Heegaard, Niels Henrik Helweg Houlind, Kim Andersen, Rikke Fredslund Brasen, Claus Lohman |
author_facet | Binderup, Helle Glud Madsen, Jonna Skov Heegaard, Niels Henrik Helweg Houlind, Kim Andersen, Rikke Fredslund Brasen, Claus Lohman |
author_sort | Binderup, Helle Glud |
collection | PubMed |
description | Numerous studies have reported a potential role for circulating microRNAs as biomarkers in a wide variety of diseases. However, there is a critical reproducibility challenge some of which might be due to differences in preanalytical and/or analytical factors. Thus, in the current study we systematically investigated the impact of selected preanalytical and analytical variables on the measured microRNA levels in plasma. Similar levels of microRNA were found in platelet-poor plasma obtained by dual compared to prolonged single centrifugation. In contrast, poor correlation was observed between measurements in standard plasma compared to platelet-poor plasma. The correlation between quantitative real-time PCR and droplet digital PCR was found to be good, contrary to TaqMan Low Density Array and single TaqMan assays where no correlation could be demonstrated. Dependent on the specific microRNA measured and the normalization strategy used, the intra- and inter-assay variation of quantitative real-time PCR were found to be 4.2–6.8% and 10.5–31.4%, respectively. Using droplet digital PCR the intra-assay variation was 4.4–20.1%, and the inter-assay variation 5.7–26.7%. Plasma preparation and microRNA purification were found to account for 39–73% of the total intra-assay variation, dependent on the microRNA measured and the normalization strategy used. In conclusion, our study highlighted the importance of reporting comprehensive methodological information when publishing, allowing others to perform validation studies where preanalytical and analytical variables as causes for divergent results can be minimized. Furthermore, if microRNAs are to become routinely used diagnostic or prognostic biomarkers, the differences in plasma microRNA levels between health and diseased subjects must exceed the high preanalytical and analytical variability. |
format | Online Article Text |
id | pubmed-6053236 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-60532362018-07-27 Quantification of microRNA levels in plasma – Impact of preanalytical and analytical conditions Binderup, Helle Glud Madsen, Jonna Skov Heegaard, Niels Henrik Helweg Houlind, Kim Andersen, Rikke Fredslund Brasen, Claus Lohman PLoS One Research Article Numerous studies have reported a potential role for circulating microRNAs as biomarkers in a wide variety of diseases. However, there is a critical reproducibility challenge some of which might be due to differences in preanalytical and/or analytical factors. Thus, in the current study we systematically investigated the impact of selected preanalytical and analytical variables on the measured microRNA levels in plasma. Similar levels of microRNA were found in platelet-poor plasma obtained by dual compared to prolonged single centrifugation. In contrast, poor correlation was observed between measurements in standard plasma compared to platelet-poor plasma. The correlation between quantitative real-time PCR and droplet digital PCR was found to be good, contrary to TaqMan Low Density Array and single TaqMan assays where no correlation could be demonstrated. Dependent on the specific microRNA measured and the normalization strategy used, the intra- and inter-assay variation of quantitative real-time PCR were found to be 4.2–6.8% and 10.5–31.4%, respectively. Using droplet digital PCR the intra-assay variation was 4.4–20.1%, and the inter-assay variation 5.7–26.7%. Plasma preparation and microRNA purification were found to account for 39–73% of the total intra-assay variation, dependent on the microRNA measured and the normalization strategy used. In conclusion, our study highlighted the importance of reporting comprehensive methodological information when publishing, allowing others to perform validation studies where preanalytical and analytical variables as causes for divergent results can be minimized. Furthermore, if microRNAs are to become routinely used diagnostic or prognostic biomarkers, the differences in plasma microRNA levels between health and diseased subjects must exceed the high preanalytical and analytical variability. Public Library of Science 2018-07-19 /pmc/articles/PMC6053236/ /pubmed/30024941 http://dx.doi.org/10.1371/journal.pone.0201069 Text en © 2018 Binderup 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 (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Binderup, Helle Glud Madsen, Jonna Skov Heegaard, Niels Henrik Helweg Houlind, Kim Andersen, Rikke Fredslund Brasen, Claus Lohman Quantification of microRNA levels in plasma – Impact of preanalytical and analytical conditions |
title | Quantification of microRNA levels in plasma – Impact of preanalytical and analytical conditions |
title_full | Quantification of microRNA levels in plasma – Impact of preanalytical and analytical conditions |
title_fullStr | Quantification of microRNA levels in plasma – Impact of preanalytical and analytical conditions |
title_full_unstemmed | Quantification of microRNA levels in plasma – Impact of preanalytical and analytical conditions |
title_short | Quantification of microRNA levels in plasma – Impact of preanalytical and analytical conditions |
title_sort | quantification of microrna levels in plasma – impact of preanalytical and analytical conditions |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6053236/ https://www.ncbi.nlm.nih.gov/pubmed/30024941 http://dx.doi.org/10.1371/journal.pone.0201069 |
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