Evaluation of reference genes for the analysis of serum miRNA in patients with prostate cancer,bladder cancer and renal cell carcinoma

Objectives: To identify an appropriate reference gene for the analysis of circulating micro‐ribonucleic acid in patients with urological malignancies. Methods: Serum from patients with prostate cancer (n = 24), bladder cancer (n = 24), renal cell carcinoma (n = 24) and control subjects (n = 48) was spiked with cel‐miR‐39, and then ribonucleic acid was isolated. Quantitative real‐time polymerase chain reaction was used to determine the levels of candidate reference genes (RNU1‐4, RNU6‐2, SNORD43, SNORD44, SNORD48, SNORA74A, miR‐let‐7a‐1, miR‐106a). Reference gene stability was determined using the NormFinder, geNorm and comparative delta‐Ct algorithm. The effect of normalization was tested with miR‐21 as the target gene, as this was previously suggested to be upregulated in cancer patients' serum. Results: Recovery of cel‐miR‐39 (mean 11.6%, range 1–56%) was similar in control subjects and cancer patients. SNORD44 and SNORD74A levels were around the detection limit of the assay and were thus omitted. All remaining candidates showed satisfying stability; SNORD43 was the most stable reference gene using all three algorithms. A combination of two genes (SNORD43, RNU1‐4) increases the stability somewhat. The level of miR‐21 was similar in cancer patients and healthy controls, irrespective of the normalization strategy. Conclusions: SNORD43 is a suitable reference gene for the analysis of circulating micro‐ribonucleic acid in patients with urological malignancies. Our study questions the suitability of miR‐21 as a biomarker for uro‐oncological patients.