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  Reference miRNAs for miRNAome Analysis of Urothelial Carcinomas

Ratert, N., Meyer, H.-A., Jung, M., Mollenkopf, H.-J., Wagner, I., Miller, K., et al. (2012). Reference miRNAs for miRNAome Analysis of Urothelial Carcinomas. PLoS ONE, 7(6): e39309. doi:10.1371/journal.pone.0039309.

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Genre: Journal Article
Alternative Title : PLoS One

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PLoS_One_2012_7_e39309.pdf (Publisher version), 280KB
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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.
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 Creators:
Ratert, Nadine, Author
Meyer, Hellmuth-Alexander, Author
Jung, Monika, Author
Mollenkopf, Hans-Joachim1, Author              
Wagner, Ina1, Author              
Miller, Kurt, Author
Kilic, Ergin, Author
Erbersdobler, Andreas, Author
Weikert, Steffen, Author
Jung, Klaus, Author
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1Core Facilities / Microarray, Max Planck Institute for Infection Biology, Max Planck Society, ou_1664141              

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 Abstract: 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.

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Language(s): eng - English
 Dates: 2012-06-20
 Publication Status: Published in print
 Pages: -
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 Table of Contents: -
 Rev. Type: Peer
 Identifiers: eDoc: 611194
ISI: 000305693200061
DOI: 10.1371/journal.pone.0039309
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Title: PLoS ONE
  Alternative Title : PLoS One
Source Genre: Journal
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Publ. Info: SAN FRANCISCO : PUBLIC LIBRARY SCIENCE
Pages: - Volume / Issue: 7 (6) Sequence Number: e39309 Start / End Page: - Identifier: ISSN: 1932-6203