Research Article Stability Assessment of Candidate Reference Genes in Urine Sediment of Prostate Cancer Patients for miRNA Applications Maria Giulia Egidi, 1 Giovanni Cochetti, 1 Gabriella Guelfi, 2 Danilo Zampini, 2 Silvana Diverio, 2 Giulia Poli, 1 and Ettore Mearini 1 1 Department of Surgical and Biomedical Sciences, Institution of Urological, Andrological Surgery and Minimally Invasive Techniques, University of Perugia, Loc. S. Andrea delle Fratte, 06156 Perugia, Italy 2 Department of Veterinary Medicine, University of Perugia, Via San Costanzo 4, 06126 Perugia, Italy Correspondence should be addressed to Giovanni Cochetti; giovannicochetti@libero.it Received 30 December 2014; Accepted 30 April 2015 Academic Editor: Ralf Lichtinghagen Copyright © 2015 Maria Giulia Egidi et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. We aimed at assessing the stability of candidate reference genes in urine sediments of men subjected to digital rectal examination for suspected prostate cancer (PCa). Two microRNAs (miR-191 and miR-25) and 1 small nucleolar RNA (SNORD48) were assayed in 35 post-DRE urine sediments of men with PCa and in 26 subjects with histologically confrmed benign prostatic hyperplasia (BPH). Te stability of candidate reference genes was assessed through BestKeeper algorithm and equivalence test. miR-200b and miR-452 were used to test for the efect of normalization on target genes. Our results proved miR-191 to be the most stable gene, showing the lowest degree of variation and the highest stability value. miR-25 and SNORD48 values fell beyond the cutofof acceptability. In conclusion, we recommend the use of miR-191 for normalization purposes in post-DRE urine sediments. 1. Introduction Prostate cancer (PCa) represents the second most common male cancer [1]. In Western countries, the main diagnostics tools for PCa diagnosis are PSA monitoring together with digital rectal examination (DRE). Although the introduction of PSA testing in clinical practice has dramatically improved the early diagnosis and consequently the treatment of local- ized disease, the real benefts of an extensive use of PSA remain controversial [2]. Since PSA is a prostate-specifc and not a cancer-specifc marker, there is an urgent need for new biomarkers able to diagnose real disease and not benign conditions which also cause an increase in serum PSA. MicroRNAs (miRNAs) represent a class of small, noncod- ing RNAs which targets complementary sites of messenger RNA (mRNA) and negatively regulates their expression [3]. In the last years, microRNAs have attracted scientifc interest upon demonstration of their alteration in response to various diseases: they have been proved to afect the crucial steps of carcinogenesis acting as oncogenes or oncosuppressors [4]. Real-time PCR is an extremely versatile tool to analyse gene expression: the selection of stably expressed reference genes is a crucial step which inevitably afects data reliability. Ofen, contrasting results are attributable to improper normalization approaches which impair the objectivity of data. For large RNAs, the stability of several reference genes has been widely reviewed [5–8] and even called into question [9–11]. For microRNAs (miRNAs), the stability of putative reference genes has not yet been completely confrmed and waits for adequate assessments in various biological matrices, though several attempts have been made so far [12–17] also for prostate cancer [18–20]. Small nucleolar RNAs (snoRNAs) [21] are quite ofen used to normalize tissue miRNA expres- sion; one such example is represented by the SNORD family, which proved to be stable in several biological matrices [22–24]. In particular, SNORD48 has been ofen used as reference gene in miRNAs expression studies on prostate cancer tissues [25–27]. Starting from these considerations, the present study was aimed at assessing the stability of miR- 25, miR-191, and SNORD48 in post-DRE urine sediments of 35 urine sediments of men undergoing radical prostatectomy for prostate cancer. Voided urine obtained immediately afer Hindawi Publishing Corporation Disease Markers Volume 2015, Article ID 973597, 6 pages http://dx.doi.org/10.1155/2015/973597