Total RNA was extracted from the bladder using Trizol
® (GIBCO/BRL Life Technologies, Inc.) according to the manufacturer's recommendations, and further purified with RNeasy columns (Qiagen, Valencia, CA, USA). All samples from the control, BCG and BCG-S1PT groups were co-processed to eliminate technical variations. Following DNase treatment (Sigma, St Louis, MO, USA), one microgram of RNA was reverse transcribed using SuperScript III reverse transcriptase (Invitrogen Corp., Carlsbad, CA, USA). The quantitative PCR (qPCR) primers used were: TNF-α (
F: CATCTTCTCAAAATTCGAGTGACAA,
R: TGGGAGTAGACAAGGTACAACCC), IL-10 (
F: GGTTGCCAAGCCTTATCGGA,
R: ACCTGCTCCACTGCCTTGCT) and GAPDH (
F: CAACTCACTCAAGATTGTCAGCAA,
R: GGCATGGACTGTGGTCATGA) (IDT, Coralville, IA, USA). qPCR was performed using an Applied Biosystems 7500 Real Time PCR System (Applied Biosystems Inc., USA). The reaction mixture contained SYBR Green PCR Master mix (Applied Biosystems), 0.5 μmol/μl l of forward and reverse primer and 1 μl of cDNA in a total volume of 10 μl. Thermocycling conditions were 50°C for 2 min., 95°C for 10 min. and 40 cycles of [95°C for 15 sec., 60°C for 1 min.]. To normalize for inefficiencies in cDNA synthesis and RNA input amounts, GAPDH mRNA expression was used. After the PCR was performed, a dissociation/melting curve was constructed in the range of 60 to 95°C. Data were analyzed using the comparative Ct method (ΔΔCt method) with the following formula: ΔCt = Ct (target) – Ct (normalized, GAPDH). The comparative ΔΔCt calculation involved finding the difference between ΔCt of the BCG and BCG-S1PT groups and the mean value of the ΔCt from control for each analyzed transcript. Fold increase in mRNA expression for the BCG groups compared to normal controls was calculated as 2
(-ΔΔCtCt) [
13].
The RNA from the bladders from Experiment II was aggregated to perform the qPCR. The RNA extracted from bladders with tumor implantation was quantified individually.