Abstract
Background/ObjectiveReverse 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.MethodsCandidate 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 FindingsBased 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.ConclusionsThe 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.
Highlights
MicroRNAs belong to a class of small noncoding RNAs of 19 to 24 nucleotides that are known to regulate signaling pathways for various cell functions
The present study provided the first systematic analysis for identifying suitable reference miRNAs for miRNA expression studies of urothelial carcinoma by Reverse transcription quantitative real-time PCR (RT-qPCR)
Several studies in addition to our own experiments have shown that the use of inappropriate reference genes in the relative quantification of gene expression can result in biased expression profiles [11,12,13]
Summary
MicroRNAs (miRNAs) belong to a class of small noncoding RNAs of 19 to 24 nucleotides that are known to regulate signaling pathways for various cell functions. To overcome experimental variations in RT-qPCR analyses (RNA isolation, cDNA synthesis, PCR runs), relative quantification of miRNAs of interest based on the normalization to reference genes is the approach of choice to prevent errors within a dataset [5]. This approach complies with normalization procedures used in mRNA expression studies and is summarized in the recent MIQE guidelines [6]. As there are no universal reference genes [14,15], it is strongly recommended that researchers test for the most suitable reference genes specific to the tissues and experimental conditions used
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