Sybr green fluorescence based assay

Total RNA was isolated using Trizol Reagent (Invitrogen, Waltham, MA), and cDNA was synthetized using a PrimeScript™ Kit (TaKaRa Bio Inc., Otsu, Japan) following the manufacturer’s instructions. qRT-PCR was performed in triplicate using a SYBR Green fluorescence-based assay (TaKaRa Bio Inc.) on a ViiA^TM7 RT-PCR system (Applied Biosystems, Carlsbad, CA). The primers used for real-time PCR were as follows: FOXC2 Forward: 5ʹ- CCTACCTGAGCGAGCAGAAT −3ʹ, Reverse: 5ʹ- ACCTTGACGAAGCACTCGTT −3ʹ; E-cadherin Forward: 5ʹ- TACGCCTGGGACTCCACCTA −3ʹ, Reverse: 5ʹ- CCAGAAACGGAGGCCTGAT −3ʹ; Vimentin Forward: 5ʹ- TGTGGATGT TTCCAAGCCTGAC −3ʹ, Reverse: 5ʹ- GAGTGGGTATCAACCAGAGGGAG −3ʹ; and GAPDH Forward: 5ʹ- CGGAGTCAACGGATTTGGTCGTAT −3ʹ, Reverse: 5ʹ- AGCCTTCTCCATGGTGGTGAAGAC −3ʹ. The relative mRNA expression values was normalized to GAPDH expression values and were calculated based on the Ct value according to the equation 2^−ΔΔCt [ΔCt=Ct (targeting gene)-Ct (GAPDH)].

The TRIzol reagent (Invitrogen, Waltham, MA) was adopted for the extraction of total RNA content, and PrimeScript™ kits were (TaKaRa Bio Inc, Otsu, Japan) utilized for the synthesis of cDNA, in accordance with the manufacturer’s protocols. A SYBR Green fluorescence-based assay (TaKaRa Bio Inc, Otsu, Japan) was adopted to perform qRT-PCR on a ViiA™ 7 RT-PCR system (Applied Biosystems, Carlsbad, CA). The primers for real-time PCR were as follows: TRPV1: forward: CCAGTCAAGCCCCACATCTT; reverse: CCAGCTCCTGGCAGTTACTC. GAPDH: forward: TGTGGGCATCAATGGATTTGG; reverse: ACACCATGATGGGGGGGGGATCAAT. TLR4: forward: CCGCTCTGGCATCATCTTCA; reverse: TCCCACTCGAGGTAGGTGTT. MyD88: forward: TGTCTCCCCTGACATGCCTA; reverse: CTGGGGGCGGAATGTTTTTGGAPDH. GAPDH was adopted as the reference control. The relative mRNA expressions were calculated using the 2-ΔΔCt method.