Tripure reagent solution

Animals (n = 8 per time point) were randomly assigned to each group. At t = 0, 45 min, and 24 h post-surgery, mice were anesthetized and euthanized, hearts were harvested and the left ventricle was dissected to maximally provide the myocardium known to be at risk in ischemic hearts. Myocardium samples were placed in RNAlater stabilizing solution (Ambion, Thermo Fisher Scientific) and stored at −80°C until use.
RNA was extracted by Tripure reagent solution (Roche), treated with Proteinase K (Qiagen) and purified by RNeasy Mini kit (Qiagen) where DNase I (Qiagen) is treated on column. RNA purity, quantity and integrity were assessed both by spectrophotometry (NanoDrop ND-1000, NanoDrop Technologies) and nanoelectrophoresis (2100 Bioanalyzer, Agilent Technologies). RNA purity: A_260/280 ∼ 1,8 and A_260/230 ∼ 2 and RNA integrity number: 8–10.

RNA was extracted from SV40-transformed H9c2 cells, mouse brain, dorsal root, and heart after homogenization using Precellys homogenizer associated with a Cryolys cooling system to ensure homogenization at 4 °C for tissues followed by Tripure reagent solution (Roche), following the manufacturer’s protocol. RNA was treated with DNAse I Amplification grade to remove any DNA contamination. cDNA synthesis was performed using PrimeScriptTM RT Reagent Kit (Perfect Real Time; Takara) as specified by the manufacturer.
TRPV1-specific primers shown in Table 1 were designed using Primer-blast software (https://www.ncbi.nlm.nih.gov/tools/primer-blast/; accessed on 8 September 2023) and tested for efficiency. All PCR reactions were run in the CFX96 C1000 system supplied by Bio-Rad. Gene amplification was performed using TB Green™ Premix Ex Taq™ (Tli RNaseH Plus, Takara Bio) under the following thermal cycling conditions: initial 95 °C for 5 min and then a 42 times repeated cycle of 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s. A melt curve was constructed in the temperature interval 65–95 °C with an increment of 0.5 °C for 5 s. Experiments were repeated for three different series. GAPDH was used as an internal control gene.