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Additional file 2: Supplementary tables, including comparison of Ct values of three genes with and without preamplification before real time qRT-PCR (Table S1), comparison of CtS values of five genes in the urine samples collected with and without digital rectal examination (DRE) (Table S2), genes in the 25-Gene Panel for prostate cancer diagnosis (Table S3), diagnostic performance of the 25-Gene Panel in GSE17951 prostate tissue specimen cohort (n = 154) (Table S4).

Additional file 1. Supplementary methods, including quantification of mRNA expression, validation of urine test without DRE, Algorithm for identification of clinically significant cancer, and statistical analysis.

Additional file 3. Supplementary figures, including box plots of biomarkers with increased gene expression levels in prostate tissue specimens from patients with prostate cancer as compared to patients with benign prostate in the GSE17951 cohort (n = 154) (Fig. S1), box plots of biomarkers with decreased gene expression levels in prostate tissue specimens from patients with prostate cancer as compared to patients with benign prostate in the GSE17951 cohort (n = 154) (Fig. S2), box plot of discriminant score F1 of the 25-Gene Panel in prostate tissue specimens from patients with prostate cancer as compared to patients with benign prostate in the GSE17951 cohort (n = 154) (Fig. S3), and receiver operating characteristic (ROC) curve of the 25-Gene Panel for PCa diagnosis in GSE17951 prostate tissue specimen cohort (n = 154) (Fig. S4).

PCR amplification was performed using Phusion DNA polymerae (Thermo Scientific) according to manufacture's recommendation. In-frame deletion in V. cholerae was performed as descibed. Briefly, a 500-bp 5-prime flanking sequence of the gene, including several nucleotides of the coding region, was PCR amplified. The two PCR products were annealed at their overlapping region and amplified by PCR to produce a single DNA fragment, using outer primers. The resulting PCR product, lacking most of the coding sequence of the gene, was digested with XbaI enzyme and ligated into a similarly digested pCVD442 suicide plasmid. pCVD442 containing mutant construct was introduced into E. coli SM10.lambda pir by electroporation. The donor, E. coli SM10-lambda pir containing the plasmid pCVD442 containing mutant construct, was used for conjugal transfer to rifampin-resistant V. cholerae A1552. A mixture containing equal volumes of the donor and recipient in LB was incubated for 6 h at 30C. The exconjugants were selected by plating the suspension onto LB plates supplemented with 100 microgram/ml of rifampin and 100 microgram/ml of carbenicillin (Cb) at 30C. After selection of the desired tranconjugants on rifampin-plus-Cb plates, they were streaked on LB plates with 10 percent sucrose at 30C. Several colonies were purified from the plates, tested for Cb sensitivity, and then analyzed for the deletion using colony PCR and DNA sequencing. Sequencing data are described here.