Int Urol Nephrol (2012) 44:761–767 Total RNA from blood samples was isolated using the PAXgene Blood RNA Kit following the manu- facturer’s instructions. One lg of RNA was used in reverse transcription (RT) reactions performed with the SuperScript II Reverse Transcriptase (Invitrogen Co., Carlsbad, CA, USA) as per the manufacturer’s protocol. Quantitative evaluation of cDNA was per- formed on the ABI 7900HT Real Time PCR system (Applied Biosystems) using either Sensimix SYBR or Probe Kit (Bioline, UK) in a 5 ll reaction mixture containing 2.5 ll 2 9 Sensimix, 2 ll cDNA (1:5 dilution) and 200 nM primers or preformulated Taq- Man Gene Expression Assays (Applied Biosystems). Q-PCR was carried out at 40 cycles, consisting of 15 s of denaturation at 95 C and hybridization for 1 min at 60 C in a 384-well reaction plate. Five serial dilutions of cDNA from normal colon tissue samples (pool of cDNAs) were used to construct the standard curves. The gene expression was regarded as continuous when ampliﬁcation signal was detected across all the samples, and relative transcripts levels were calculated using the DDCT method. For the transcripts with dichotomous expression (positive/negative signal of expression), we calculated the percent of positive results for a group of patients. Oligonucleotide primers for the analyzed gene transcripts, used for Sybr Green I Dye Assays, were either designed using Primer Express Software (Applied Biosystems) or taken from qPrimer Depot database [25] and are listed in Table 1. The results of gene expression levels were analyzed with the Mann–Whitney U test, and differences between numbers of samples with detectable signal were analyzed using the Fisher’s exact test. P values of less than 0.01 were considered signiﬁcant. Results Selection of potential biomarkers Gene expression was analyzed using qRT-PCR with 40 ampliﬁcation cycles using either pairs of designed primers with SYBR Green chemistry or TaqMan Gene Expression Assay. To determine the potential speci- ﬁcity of both literature- and microarray-selected genes (listed in the ‘Methods’ section), analyses of expres- sion were ﬁrstly conducted on RNA isolated from blood samples collected from two groups of control subjects: 17 controls recruited from the laboratory 763 staff and 15 controls from whom blood samples were taken immediately before colonoscopy. As shown in Table 1, while the expression of 8 of 14 studied genes was found for most control blood samples, detectable signals of EGFR, COL1A1 and AGR2 could be determined for only a few control samples. Expression of TERT, TP53, SNX16 and VEGFA signiﬁcantly varied between series of PCR assays due to very low signal level (detectable above 37 cycles of ampliﬁcation), which led to low repro- ducibility of results. In addition, qRT-PCR analyses revealed signiﬁcant differences in expression of two genes between the two control groups. The detectable signal of UPK2 mRNA was found in one of 17 control blood samples obtained from laboratory staff and in 14 of 15 blood samples collected immediately before colonoscopy (P = 8,551E-07). Also, the relative levels of KIT expression were signiﬁcantly higher (P = 4,55E-06) in blood samples collected in healthy controls before colonoscopy compared to laboratory staff controls. Biomarkers differentiating between healthy controls and bladder cancer patients Then, we compared gene expression estimated in blood samples collected from BCa patients a day before TURBT and from control individuals. The expression level of CD47 was signiﬁcantly higher in blood samples collected from BCa patients compared to both control groups (P = 1.19E-12 and P = 1.83E-12, respectively), and the expression of KIT was signiﬁcantly higher compared to controls selected only from laboratory staff (P = 3.68E-03). Among genes with expression detected in a limited number of blood samples, mRNAs of EGFR and COL1A1 were found signiﬁcantly more frequently in cancer patient samples than in controls (P = 0.000605 and P = 0.006737, respectively). Expression of AGR2 was not detectable in blood samples from cancer patients. Expression of all other genes selected for this study did not differentiate the bladder cancer patient group from healthy controls. Histological grading is an important prognostic factor for bladder tumors, especially for prediction of progression [1, 26, 27]. Superﬁcial low-grade cancers frequently recur, but they exhibit a minimal risk for progression, while high-grade, locally advanced tumors often metastasize and therefore, result in poor 123