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.