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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...

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Autores principales: 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
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2017
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.
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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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