First strand synthesis kit

Total RNA was extracted from the frozen bladder tissues using Trizol Reagent(Tiangen Biotech Co.,Ltd. Beijing, China) in accordance with the vendor’s instructions. First strand cDNA was synthesized using Oligo(dT)₁₅ primers in a first strand synthesis kit (Promega Tech, Co., Ltd, USA). Real-time PCR was conducted using PCR Master Mix (Promega Tech, Co., Ltd, USA) according to the vendor’s recommendations. Total RNA was reverse-transcribed and subjected to PCR as follows: 95°C for 2minutes followed by 30 cycles, each cycle comprising of 95°C for 30s, 56°C for 30s and 72°C for 45s,and a final extension at 72°C for 7minutes.The primer sequences amplified were as follows.
Collagen 3: sense 5’-TGATGGGATCCAATGAGGGAGA-3’, anti-sense: 5’-GAG TCT CATGGCCTTGCGTGTTT-3’
Primer sequences for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were 5’-CGTCATTGACCTCAACTACATG-3’ (sense) and 5’-CTTCTCC ACGGTGGTGAAGAC-3’ (anti-sense) as an internal standard. Reverse-transcribed PCR products were detected by 2% agarose gel electrophoresis. Expression levels of target mRNA were represented by ratios of TGFβ1/ GAPDH, Smad2/ GAPDH, Smad3/ GAPDH, α-SMA/ GAPDH, Collagen1/ GAPDH, Collagen3/ GAPDH mRNA and showed as the mean ±S.D.

The Total RNA plus kit (Norgen Biotek Corp, Canada) was used to isolate RNA from cell and tissues according to the manufacturer’s instructions. Following stimulation, the medium was aspirated and cell monolayers were washed twice with ice-cold PBS and lysed directly in buffer RL. Mouse tissues were disaggregated in ice-cold buffer RL using a Precellys 24 tissue homogenizer set at 2 cycles a 6500 rpm for 20 s with a 30 s interval at 4 °C. Total RNA was extracted and subjected to DNase-1 digestion and ~ 1 μg was reverse transcribed with oligo-dT₁₈ primers using the First Strand Synthesis Kit (Promega) for 1 h at 48 °C. Real-time qPCR was performed using a Rotagene RG-3000 (Qiagen) under the following amplification conditions: 95 °C for 10 min, followed by 45 cycles of 95 °C, 10 s; 58 °C, 10 s; 72 °C, 20 s. cDNA samples were amplified in triplicate using SensiMix SYBR green (Bioline) and primers specific for human and mouse PlGF, FOXO1, FOXO3A, VEGF and β-actin (Table S1). PCR products were analysed by 2% agarose gel electrophoresis, sequenced, and post-run melt curve analysis performed to ensure specificity of reactions. The mean threshold cycle (Ct) for each sample was normalised to β-actin levels and fold changes to the experimental control calculated using the ^ΔΔCt method.