Real-time quantitative RT-PCR for TRPA1 mRNA in T1−T4 DRG.

Real-time quantitative RT-PCR was performed to determine the expression of TRPA1 mRNA in resected T1−T4 DRG samples. Tissues were lysed, homogenized, resuspended in PBS, and then incubated with 1 µl TRIzol reagent (Invitrogen, Carlsbad, CA). RNA concentration and quality were measured using a Nanodrop (ThermoFisher Scientific).

Reverse transcription was performed using the QuantiTect RT kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Then, 1 µl of each reverse transcribed cDNA product was used for the quantitative PCR. PCR master mix was prepared using 5 µl SYBR green (Life Technologies), 3.8 µl RNase-free water, 0.1 µl forward primer (TRPA1 or β-actin, Integrated DNA Technologies, Coralville, IA), and 0.1 µl reverse primer (TRPA1 or β-actin) per 10-µl reaction. The relative expression of TRPA1 mRNA was normalized against β-actin mRNA using the ΔΔC_t method (where Ct is threshold cycle) and expressed as fold changes (2−ΔΔCt). The following primer sequences were used for TRPA1: forward 5′-CTGTGAAGCGCTGAATGTAATG-3′ and reverse 5′-GCTCCTTGGCTGAGAAGAAA-3′. The following primer sequences were used for β-actin: forward 5′-ACAGGATGCAGAAGGAGATTAC-3′ and reverse 5′-ACAGTGAGGCCAGGATAGA-3′.