2.12. RNA Extraction and Real-Time Polymerase Chain Reaction (RT-PCR)

To evaluate TNF-α and COX-2 genes, part of the heart tissue was considered for molecular tests. The heart samples were immediately transferred to microtubes of DNase & RNase free, and the samples were stored in a -20°C freezer overnight; then, the liquid was removed and tissues were transferred to a -80°C freezer and stored until RNA extraction. Then, the total RNA was extracted based on the total RNA extraction kit protocol (Pars Tous, Mashhad, Iran). Also, cDNA was synthesized according to the manufacturer's protocol (Pars Tous, Mashhad, Iran). qRT-PCR was conducted using an ABI Step One Plus Real-Time PCR System (Applied Biosystem, USA) with primer sets for COX-2 and TNF-α as target genes and GAPDH as a housekeeping gene. 10 μL real-time PCR reaction mixture comprised of 1 μL cDNA, 6.25 μL SYBR-Green (Amplicon high Rox master mix, Denmark), 2.25 μL nuclease-free water, and 0.25 μL of 10 pmol of each primer (Robin Teb Gostar, Tehran, Iran).

According to Kirkpatrick et al. [27], conditions for the reverse transcription step were 25°C for 10 min, 37°C for 60 min, and 85°C for 5 min. The polymerase chain reaction was carried out by holding temperature for 15 min at 95°C and after that 40 cycles of 15 s at 95°C, 30 s at 62°C, and 30 s at 72°C followed by melting curve temperature steps. The COX-2 and TNF-α primers are represented in Table 1.

Primer sequences of COX-2, TNF-α, and the housekeeping genes.