Total RNA was isolated from normal peritumoral kidney tissues and RCC samples with an RNeasy® Mini Kit and RNase-free DNase set (Qiagen NV, Venlo, the Netherlands), and approximately 0.5 μg of extracted RNA was reverse transcribed using a First Strand cDNA Synthesis Kit for RT-PCR (Hoffman-La Roche Ltd., Basel, Switzerland) with random primers.
Real-time RT-PCR assays were performed with a Smart-Cycler® System (Cepheid, Sunnyvale, CA, USA) using SYBR® Green-I as the fluorogenic dye (Molecular Probes, Eugene, OR, USA). Two microliters of the complementary DNA were added into a 25 μL reaction system of Ex-taq® RT-PCR version (TaKaRa, Otsu, Japan) with 0.2 μM of each pair of gene-specific primers and then subjected to 45 polymerase chain reaction (PCR) cycles. The primer sequences of TRPV1 (GenBank® no. NM-080705.3) were as follows: forward, 5′-TCA ACA AGA TCG CAC AGG AG-3′; reverse, 5′-GCC TGA AAC TCT GCT TGA CC-3′.
The thermal program was 5 seconds at 95°C for denaturation and 20 seconds at 60°C for annealing and elongation. The mRNA expression level was normalized as the ratio to β-actin expression (forward, 5′-GGA CTT CGA GCA AGA GAT GG-3′; reverse, 5′-AGC ACT GTG TTG GCG TAC AG-3′) in each sample. Amplified PCR products were electrophoresed on 2.5% agarose gel for verification.
Real-time RT-PCR assays were performed with a Smart-Cycler® System (Cepheid, Sunnyvale, CA, USA) using SYBR® Green-I as the fluorogenic dye (Molecular Probes, Eugene, OR, USA). Two microliters of the complementary DNA were added into a 25 μL reaction system of Ex-taq® RT-PCR version (TaKaRa, Otsu, Japan) with 0.2 μM of each pair of gene-specific primers and then subjected to 45 polymerase chain reaction (PCR) cycles. The primer sequences of TRPV1 (GenBank® no. NM-080705.3) were as follows: forward, 5′-TCA ACA AGA TCG CAC AGG AG-3′; reverse, 5′-GCC TGA AAC TCT GCT TGA CC-3′.
The thermal program was 5 seconds at 95°C for denaturation and 20 seconds at 60°C for annealing and elongation. The mRNA expression level was normalized as the ratio to β-actin expression (forward, 5′-GGA CTT CGA GCA AGA GAT GG-3′; reverse, 5′-AGC ACT GTG TTG GCG TAC AG-3′) in each sample. Amplified PCR products were electrophoresed on 2.5% agarose gel for verification.