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          Institute: MPI für Infektionsbiologie     Collection: Core Facilites     Display Documents



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ID: 611194.0, MPI für Infektionsbiologie / Core Facilites
Reference miRNAs for miRNAome Analysis of Urothelial Carcinomas
Authors:Ratert, Nadine; Meyer, Hellmuth-Alexander; Jung, Monika; Mollenkopf, Hans-Joachim; Wagner, Ina; Miller, Kurt; Kilic, Ergin; Erbersdobler, Andreas; Weikert, Steffen; Jung, Klaus
Language:English
Date of Publication (YYYY-MM-DD):2012-06-20
Title of Journal:PLoS ONE
Journal Abbrev.:PLoS One
Volume:7
Issue / Number:6
Sequence Number of Article:e39309
Copyright:Copyright Ratert et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Review Status:Peer-review
Audience:Experts Only
Abstract / Description:Background/Objective: 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. Methods: 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. Principal Findings: 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. Conclusions: 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.
External Publication Status:published
Document Type:Article
Communicated by:Beate Löhr
Affiliations:MPI für Infektionsbiologie/Core Facilities
External Affiliations:[Ratert, Nadine; Meyer, Hellmuth-Alexander; Jung, Monika; Miller, Kurt; Weikert, Steffen; Jung, Klaus] Univ Hosp Charite, Dept Urol, Berlin, Germany.; [Ratert, Nadine; Jung, Klaus] Berlin Inst Urol Res, Berlin, Germany.; [Meyer, Hellmuth-Alexander] Univ Hosp Charite, Inst Physiol, Berlin, Germany.; [Kilic, Ergin] Univ Hosp Charite, Inst Pathol, Berlin, Germany.; [Erbersdobler, Andreas] Univ Rostock, Inst Pathol, Rostock, Germany.
Identifiers:ISI:000305693200061 [ID No:1]
ISSN:1932-6203 [ID No:2]
DOI:10.1371/journal.pone.0039309 [ID No:3]
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