Reference genes for the relative quantification of microRNAs in renal cell carcinomas and their metastases.

Abstract

To obtain accurate results in miRNA expression changes between different sample sets using real-time quantitative polymerase chain reaction (RT-qPCR) analyses, normalization to reference genes that are stably expressed across the sample sets is generally used. A literature search of miRNA expression studies in renal cell carcinoma (RCC) proved that non-miRNAs such as small RNAs or mRNAs have most frequently been used without preceding validation of their suitability. In this study, the most stably expressed miRNAs were ascertained from microarray-based data of miRNA expression in nonmalignant and malignant samples from clear cell RCC and from corresponding distant RCC metastases using the geNorm and NormFinder algorithms. Validation experiments with RT-qPCR were performed for the four best-ranked miRNAs (miR-28, miR-103, miR-106a, miR-151) together with the small RNU6B, RNU44, and RNU48 mostly described in literature. miR-28, miR-103, miR-106a, and RNU48 were proved as the most stably expressed genes. miR-28 is recommended as normalizer if only a single reference gene can be used, while the combinations of miR-28 and miR-103 or of miR-28, miR-103, and miR-106a, respectively, are preferred. RNU6B most frequently used as normalizer in miRNA expression studies should be abandoned in order to avoid misleading results.

• Authors: Zofia Wotschofsky, Helmuth-Alexander Meyer, Monika Jung, Annika Fendler, Ina Wagner, Carsten Stephan, Jonas Busch, Andreas Erbersdobler, Alexander C Disch, Hans-Joachim Mollenkopf, Klaus Jung
• Journal: Analytical biochemistry (Anal Biochem) Vol. 417 Issue 2 Pg. 233-41 (Oct 15 2011) ISSN: 1096-0309 [Electronic] United States
• PMID: 21741950 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2011 Elsevier Inc. All rights reserved.

Chemical References

• MIRN103 microRNA, human
• MIRN106 microRNA, human
• MicroRNAs
• RNA, Small Nuclear
• U6 small nuclear RNA

Topics

• Carcinoma, Renal Cell (genetics, secondary)
• Gene Expression Regulation, Neoplastic
• Genes, Neoplasm
• Humans
• Kidney Neoplasms (genetics, pathology)
• MicroRNAs (analysis, genetics)
• Neoplasm Metastasis
• Oligonucleotide Array Sequence Analysis
• RNA, Small Nuclear (genetics)
• Real-Time Polymerase Chain Reaction
• Reference Standards