Journal
ANALYTICAL BIOCHEMISTRY
Volume 417, Issue 2, Pages 233-241Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2011.06.009
Keywords
MicroRNAs; Reference genes; Renal cell carcinoma; RT-qPCR
Funding
- Foundation for Urological Research [BFIU_2010]
- SONNENFELD-Stiftung [89838210]
- Federal Ministry of Education and Research (MedSys)
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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. (C) 2011 Elsevier Inc. All rights reserved.
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