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Defining quantification methods and optimizing protocols for microarray hybridization of circulating microRNAs
MicroRNAs (miRNAs) have emerged as promising biomarkers of disease. Their potential use in clinical practice requires standardized protocols with very low miRNA concentrations, particularly in plasma samples. Here we tested the most appropriate method for miRNA quantification and validated the perfo...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5552704/ https://www.ncbi.nlm.nih.gov/pubmed/28798363 http://dx.doi.org/10.1038/s41598-017-08134-3 |
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author | Garcia-Elias, Anna Alloza, Leonor Puigdecanet, Eulàlia Nonell, Lara Tajes, Marta Curado, Joao Enjuanes, Cristina Díaz, Oscar Bruguera, Jordi Martí-Almor, Julio Comín-Colet, Josep Benito, Begoña |
author_facet | Garcia-Elias, Anna Alloza, Leonor Puigdecanet, Eulàlia Nonell, Lara Tajes, Marta Curado, Joao Enjuanes, Cristina Díaz, Oscar Bruguera, Jordi Martí-Almor, Julio Comín-Colet, Josep Benito, Begoña |
author_sort | Garcia-Elias, Anna |
collection | PubMed |
description | MicroRNAs (miRNAs) have emerged as promising biomarkers of disease. Their potential use in clinical practice requires standardized protocols with very low miRNA concentrations, particularly in plasma samples. Here we tested the most appropriate method for miRNA quantification and validated the performance of a hybridization platform using lower amounts of starting RNA. miRNAs isolated from human plasma and from a reference sample were quantified using four platforms and profiled with hybridization arrays and RNA sequencing (RNA-seq). Our results indicate that the Infinite(®) 200 PRO Nanoquant and Nanodrop 2000 spectrophotometers magnified the miRNA concentration by detecting contaminants, proteins, and other forms of RNA. The Agilent 2100 Bioanalyzer PicoChip and SmallChip gave valuable information on RNA profile but were not a reliable quantification method for plasma samples. The Qubit(®) 2.0 Fluorometer provided the most accurate quantification of miRNA content, although RNA-seq confirmed that only ~58% of small RNAs in plasma are true miRNAs. On the other hand, reducing the starting RNA to 70% of the recommended amount for miRNA profiling with arrays yielded results comparable to those obtained with the full amount, whereas a 50% reduction did not. These findings provide important clues for miRNA determination in human plasma samples. |
format | Online Article Text |
id | pubmed-5552704 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-55527042017-08-14 Defining quantification methods and optimizing protocols for microarray hybridization of circulating microRNAs Garcia-Elias, Anna Alloza, Leonor Puigdecanet, Eulàlia Nonell, Lara Tajes, Marta Curado, Joao Enjuanes, Cristina Díaz, Oscar Bruguera, Jordi Martí-Almor, Julio Comín-Colet, Josep Benito, Begoña Sci Rep Article MicroRNAs (miRNAs) have emerged as promising biomarkers of disease. Their potential use in clinical practice requires standardized protocols with very low miRNA concentrations, particularly in plasma samples. Here we tested the most appropriate method for miRNA quantification and validated the performance of a hybridization platform using lower amounts of starting RNA. miRNAs isolated from human plasma and from a reference sample were quantified using four platforms and profiled with hybridization arrays and RNA sequencing (RNA-seq). Our results indicate that the Infinite(®) 200 PRO Nanoquant and Nanodrop 2000 spectrophotometers magnified the miRNA concentration by detecting contaminants, proteins, and other forms of RNA. The Agilent 2100 Bioanalyzer PicoChip and SmallChip gave valuable information on RNA profile but were not a reliable quantification method for plasma samples. The Qubit(®) 2.0 Fluorometer provided the most accurate quantification of miRNA content, although RNA-seq confirmed that only ~58% of small RNAs in plasma are true miRNAs. On the other hand, reducing the starting RNA to 70% of the recommended amount for miRNA profiling with arrays yielded results comparable to those obtained with the full amount, whereas a 50% reduction did not. These findings provide important clues for miRNA determination in human plasma samples. Nature Publishing Group UK 2017-08-10 /pmc/articles/PMC5552704/ /pubmed/28798363 http://dx.doi.org/10.1038/s41598-017-08134-3 Text en © The Author(s) 2017 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 Garcia-Elias, Anna Alloza, Leonor Puigdecanet, Eulàlia Nonell, Lara Tajes, Marta Curado, Joao Enjuanes, Cristina Díaz, Oscar Bruguera, Jordi Martí-Almor, Julio Comín-Colet, Josep Benito, Begoña Defining quantification methods and optimizing protocols for microarray hybridization of circulating microRNAs |
title | Defining quantification methods and optimizing protocols for microarray hybridization of circulating microRNAs |
title_full | Defining quantification methods and optimizing protocols for microarray hybridization of circulating microRNAs |
title_fullStr | Defining quantification methods and optimizing protocols for microarray hybridization of circulating microRNAs |
title_full_unstemmed | Defining quantification methods and optimizing protocols for microarray hybridization of circulating microRNAs |
title_short | Defining quantification methods and optimizing protocols for microarray hybridization of circulating microRNAs |
title_sort | defining quantification methods and optimizing protocols for microarray hybridization of circulating micrornas |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5552704/ https://www.ncbi.nlm.nih.gov/pubmed/28798363 http://dx.doi.org/10.1038/s41598-017-08134-3 |
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