title: Reference miRNAs for miRNAome analysis of urothelial carcinomas.
abstract: 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.
  
  
  RESULTS
  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.
authors: Department of Urology, University Hospital Charité, Berlin, Germany.
authors: Nadine
authors: N
authors: Ratert
authors: Hellmuth-Alexander
authors: HA
authors: Meyer
authors: Monika
authors: M
authors: Jung
authors: Hans-Joachim
authors: HJ
authors: Mollenkopf
authors: Ina
authors: I
authors: Wagner
authors: Kurt
authors: K
authors: Miller
authors: Ergin
authors: E
authors: Kilic
authors: Andreas
authors: A
authors: Erbersdobler
authors: Steffen
authors: S
authors: Weikert
authors: Klaus
authors: K
authors: Jung
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000235:genetics*; D008875:Middle Aged; D060888:Real-Time Polymerase Chain Reaction; D001749:Urinary Bladder Neoplasms / Q000235:genetics*
chemical_list: D035683:MicroRNAs