Bladder Tissue RNA Isolation and RT-qPCR

Using the RNeasy-Mini Kit (Qiagen Inc., Valencia, CA, USA), total RNA from bladder tissues was isolated, and the DNA-free Kit (Applied Biosystems, Foster City, CA, USA) was used to remove genomic DNA. Using Taqman reverse transcription reagents (Applied Biosystems), mRNA (400 ng) was reverse transcribed per manufacturer’s instructions. Quantitative assessment of the target genes’ expression levels was performed using real-time quantitative PCR (RQ-PCR) with a PikoReal™ Real-Time PCR System (Thermo Scientific) with iQ™ SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA, USA), as described previously^{39 (link),40 (link)}. Gene expression data were quantified using duplicated RQ-PCR assays (n = 10) from five independent animals.