Abstract: Objective  To identify the most stable reference genes for the quantification of serum miRNA in patients with bladder cancer. Methods  A total of 100 muscle-invasive bladder cancer (MIBC) patients, 105 non-muscle-invasive bladder cancer (NMIBC) patients, and 139 healthy controls were included in our study. MiSeq sequencing was performed, then the expressions of selected miRNAs by sequencing and U6 were examined by real time quantitative polymerase chain reaction (qRT-PCR), and the stability was analyzed with geNorm and NormFinder. The availability of the most stable reference genes was validated again in a new cohort. Furthermore, the miR-148b-3p was selected as a target gene to determine the effect of candidate normalizers on miRNA quantification. Results  After confirmation by qRT-PCR, 5 genes (hsa-miR-193a-5p, hsa-miR-16-5p, U6, hsa-miR-191-5p and hsa-let-7d-3p) were selected as candidate reference genes. The analysis and validation Results   showed that hsa-miR-193a-5p was the most stably expressed, and the combination of reference genes hsa-miR-193a-5p and hsa-miR-16-5p was suggested. Furthermore, the expression of miR-148b-3p in bladder cancer serum was significantly up-regulated and could be valuable in the diagnosis of bladder cancer. Conclusion  Hsa-miR-193a-5p is the most stable and normalization of miRNA data using a combination of hsa-miR-193a-5p and hsa-miR-16-5p as reference genes may produce reliable and accurate Results   for serum miRNAs study in bladder cancer.

Key words: qRT-PCR, Bladder cancer, microRNA, Reference genes