Reference miRNAs for miRNAome analysis of urothelial carcinomas.

Reference miRNAs for miRNAome analysis of urothelial carcinomas.

<h4>Background/objective</h4>Reverse transcription quantitative real-time PCR (RT-qPCR) is widely used in microRNA (miRNA) expression studies on cancer. To compensate for the analytical variability produced by the multiple steps of the method, relative quantification of the measured miRN...

Descripción completa

Guardado en:
Detalles Bibliográficos
Autores principales: Nadine Ratert, Hellmuth-Alexander Meyer, Monika Jung, Hans-Joachim Mollenkopf, Ina Wagner, Kurt Miller, Ergin Kilic, Andreas Erbersdobler, Steffen Weikert, Klaus Jung
Formato: article
Lenguaje:EN
Publicado: Public Library of Science (PLoS) 2012
Materias:
Medicine
R
Science
Q
Acceso en línea:https://doaj.org/article/3cf2a028b3454d0aa4cb56b48aa604b4
Etiquetas: Agregar Etiqueta
Sin Etiquetas, Sea el primero en etiquetar este registro!
id oai:doaj.org-article:3cf2a028b3454d0aa4cb56b48aa604b4
record_format dspace
spelling oai:doaj.org-article:3cf2a028b3454d0aa4cb56b48aa604b42021-11-18T07:14:50ZReference miRNAs for miRNAome analysis of urothelial carcinomas.1932-620310.1371/journal.pone.0039309https://doaj.org/article/3cf2a028b3454d0aa4cb56b48aa604b42012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22745731/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background/objective</h4>Reverse transcription quantitative real-time PCR (RT-qPCR) is widely used in microRNA (miRNA) expression studies on cancer. To compensate for the analytical variability produced by the multiple steps of the method, relative quantification of the measured miRNAs is required, which is based on normalization to endogenous reference genes. No study has been performed so far on reference miRNAs for normalization of miRNA expression in urothelial carcinoma. The aim of this study was to identify suitable reference miRNAs for miRNA expression studies by RT-qPCR in urothelial carcinoma.<h4>Methods</h4>Candidate reference miRNAs were selected from 24 urothelial carcinoma and normal bladder tissue samples by miRNA microarrays. The usefulness of these candidate reference miRNAs together with the commonly for normalization purposes used small nuclear RNAs RNU6B, RNU48, and Z30 were thereafter validated by RT-qPCR in 58 tissue samples and analyzed by the algorithms geNorm, NormFinder, and BestKeeper.<h4>Principal findings</h4>Based on the miRNA microarray data, a total of 16 miRNAs were identified as putative reference genes. After validation by RT-qPCR, miR-101, miR-125a-5p, miR-148b, miR-151-5p, miR-181a, miR-181b, miR-29c, miR-324-3p, miR-424, miR-874, RNU6B, RNU48, and Z30 were used for geNorm, NormFinder, and BestKeeper analyses that gave different combinations of recommended reference genes for normalization.<h4>Conclusions</h4>The present study provided the first systematic analysis for identifying suitable reference miRNAs for miRNA expression studies of urothelial carcinoma by RT-qPCR. Different combinations of reference genes resulted in reliable expression data for both strongly and less strongly altered miRNAs. Notably, RNU6B, which is the most frequently used reference gene for miRNA studies, gave inaccurate normalization. The combination of four (miR-101, miR-125a-5p, miR-148b, and miR-151-5p) or three (miR-148b, miR-181b, and miR-874,) reference miRNAs is recommended for normalization.Nadine RatertHellmuth-Alexander MeyerMonika JungHans-Joachim MollenkopfIna WagnerKurt MillerErgin KilicAndreas ErbersdoblerSteffen WeikertKlaus JungPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 6, p e39309 (2012)
institution DOAJ
collection DOAJ
language EN
topic Medicine
R
Science
Q
spellingShingle Medicine
R
Science
Q
Nadine Ratert
Hellmuth-Alexander Meyer
Monika Jung
Hans-Joachim Mollenkopf
Ina Wagner
Kurt Miller
Ergin Kilic
Andreas Erbersdobler
Steffen Weikert
Klaus Jung
Reference miRNAs for miRNAome analysis of urothelial carcinomas.
description <h4>Background/objective</h4>Reverse transcription quantitative real-time PCR (RT-qPCR) is widely used in microRNA (miRNA) expression studies on cancer. To compensate for the analytical variability produced by the multiple steps of the method, relative quantification of the measured miRNAs is required, which is based on normalization to endogenous reference genes. No study has been performed so far on reference miRNAs for normalization of miRNA expression in urothelial carcinoma. The aim of this study was to identify suitable reference miRNAs for miRNA expression studies by RT-qPCR in urothelial carcinoma.<h4>Methods</h4>Candidate reference miRNAs were selected from 24 urothelial carcinoma and normal bladder tissue samples by miRNA microarrays. The usefulness of these candidate reference miRNAs together with the commonly for normalization purposes used small nuclear RNAs RNU6B, RNU48, and Z30 were thereafter validated by RT-qPCR in 58 tissue samples and analyzed by the algorithms geNorm, NormFinder, and BestKeeper.<h4>Principal findings</h4>Based on the miRNA microarray data, a total of 16 miRNAs were identified as putative reference genes. After validation by RT-qPCR, miR-101, miR-125a-5p, miR-148b, miR-151-5p, miR-181a, miR-181b, miR-29c, miR-324-3p, miR-424, miR-874, RNU6B, RNU48, and Z30 were used for geNorm, NormFinder, and BestKeeper analyses that gave different combinations of recommended reference genes for normalization.<h4>Conclusions</h4>The present study provided the first systematic analysis for identifying suitable reference miRNAs for miRNA expression studies of urothelial carcinoma by RT-qPCR. Different combinations of reference genes resulted in reliable expression data for both strongly and less strongly altered miRNAs. Notably, RNU6B, which is the most frequently used reference gene for miRNA studies, gave inaccurate normalization. The combination of four (miR-101, miR-125a-5p, miR-148b, and miR-151-5p) or three (miR-148b, miR-181b, and miR-874,) reference miRNAs is recommended for normalization.
format article
author Nadine Ratert
Hellmuth-Alexander Meyer
Monika Jung
Hans-Joachim Mollenkopf
Ina Wagner
Kurt Miller
Ergin Kilic
Andreas Erbersdobler
Steffen Weikert
Klaus Jung
author_facet Nadine Ratert
Hellmuth-Alexander Meyer
Monika Jung
Hans-Joachim Mollenkopf
Ina Wagner
Kurt Miller
Ergin Kilic
Andreas Erbersdobler
Steffen Weikert
Klaus Jung
author_sort Nadine Ratert
title Reference miRNAs for miRNAome analysis of urothelial carcinomas.
title_short Reference miRNAs for miRNAome analysis of urothelial carcinomas.
title_full Reference miRNAs for miRNAome analysis of urothelial carcinomas.
title_fullStr Reference miRNAs for miRNAome analysis of urothelial carcinomas.
title_full_unstemmed Reference miRNAs for miRNAome analysis of urothelial carcinomas.
title_sort reference mirnas for mirnaome analysis of urothelial carcinomas.
publisher Public Library of Science (PLoS)
publishDate 2012
url https://doaj.org/article/3cf2a028b3454d0aa4cb56b48aa604b4
work_keys_str_mv AT nadineratert referencemirnasformirnaomeanalysisofurothelialcarcinomas
AT hellmuthalexandermeyer referencemirnasformirnaomeanalysisofurothelialcarcinomas
AT monikajung referencemirnasformirnaomeanalysisofurothelialcarcinomas
AT hansjoachimmollenkopf referencemirnasformirnaomeanalysisofurothelialcarcinomas
AT inawagner referencemirnasformirnaomeanalysisofurothelialcarcinomas
AT kurtmiller referencemirnasformirnaomeanalysisofurothelialcarcinomas
AT erginkilic referencemirnasformirnaomeanalysisofurothelialcarcinomas
AT andreaserbersdobler referencemirnasformirnaomeanalysisofurothelialcarcinomas
AT steffenweikert referencemirnasformirnaomeanalysisofurothelialcarcinomas
AT klausjung referencemirnasformirnaomeanalysisofurothelialcarcinomas
_version_ 1718423720336293888

Ejemplares similares