Defining quantification methods and optimizing protocols for microarray hybridization of circulating microRNAs

Abstract 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...

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Autores principales:	Anna Garcia-Elias, Leonor Alloza, Eulàlia Puigdecanet, Lara Nonell, Marta Tajes, Joao Curado, Cristina Enjuanes, Oscar Díaz, Jordi Bruguera, Julio Martí-Almor, Josep Comín-Colet, Begoña Benito
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/b4e3539c8ab74c0ca848f212ca0dab8a
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spelling	oai:doaj.org-article:b4e3539c8ab74c0ca848f212ca0dab8a2021-12-02T16:06:32ZDefining quantification methods and optimizing protocols for microarray hybridization of circulating microRNAs10.1038/s41598-017-08134-32045-2322https://doaj.org/article/b4e3539c8ab74c0ca848f212ca0dab8a2017-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-08134-3https://doaj.org/toc/2045-2322Abstract 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.Anna Garcia-EliasLeonor AllozaEulàlia PuigdecanetLara NonellMarta TajesJoao CuradoCristina EnjuanesOscar DíazJordi BrugueraJulio Martí-AlmorJosep Comín-ColetBegoña BenitoNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-14 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Anna Garcia-Elias Leonor Alloza Eulàlia Puigdecanet Lara Nonell Marta Tajes Joao Curado Cristina Enjuanes Oscar Díaz Jordi Bruguera Julio Martí-Almor Josep Comín-Colet Begoña Benito Defining quantification methods and optimizing protocols for microarray hybridization of circulating microRNAs
description	Abstract 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	article
author	Anna Garcia-Elias Leonor Alloza Eulàlia Puigdecanet Lara Nonell Marta Tajes Joao Curado Cristina Enjuanes Oscar Díaz Jordi Bruguera Julio Martí-Almor Josep Comín-Colet Begoña Benito
author_facet	Anna Garcia-Elias Leonor Alloza Eulàlia Puigdecanet Lara Nonell Marta Tajes Joao Curado Cristina Enjuanes Oscar Díaz Jordi Bruguera Julio Martí-Almor Josep Comín-Colet Begoña Benito
author_sort	Anna Garcia-Elias
title	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_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_sort	defining quantification methods and optimizing protocols for microarray hybridization of circulating micrornas
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/b4e3539c8ab74c0ca848f212ca0dab8a
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_version_	1718384984247500800

Defining quantification methods and optimizing protocols for microarray hybridization of circulating microRNAs

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