Cdna synthesis kit

Total RNA extraction kit (Bio E-Technology, China) was used for extraction of total RNA from heart tissue, purity and concentration of mRNA were estimated at A260/A280 nm using Nanodrop spectrophotometer (Analytik Jena, Italy). Complementary DNA (cDNA) was synthesized from extracted mRNA using cDNA synthesis kit (Origene™ Technologies, USA). The cDNA was amplified using SensiMix SYBR Master Mix (Origene™ Technologies, USA). Primer sequences were designed by Primer 3 plus Program (version 2.0) and purchased from Biosearch Technologies Co., (California, USA). Primer sequences of ATG-5: F (5′-GGCATGCTTCCCTAACTTGA-3′) & R (5′-CCCACCCATCCAAGAGTACA-3′), Beclin-1 (ATG-6): F (5`-TGGATCTGGACCAGGGGTCCTTGCG-3’) & R (5′-GTTTCGCCTGGGCTGTGGTAAGTAA-3`), TNF-α: F (5′-CTTCTCCTTCCT GAT CGT GG-3′) & R (5′-GCT GGT TATCTCTCAGCT CCA-3′) and GAPDH (Control Gene): F (5′-GAAGGT GAA GGTCGG AGT-3′) & R (5′-GAAGATGGT GATGGG ATT TC-3′). Each sample was analyzed by qRT-PCR system Pikoreal 5100 (Thermo Fisher Scientific Co., Finland), normalized to the level of the reference gene, and threshold cycle (C_t) values were calculated. The relative gene expression level was calculated by using 2^−ΔCt formula [20 (link)].

The extraction of RNA was performed with the miRNAeasy Mini Kit (Qiagen). Reverse transcription of RNA was carried out using the “cDNA Synthesis kit” (OriGene). The PCR was performed with a master mix, containing SYBR green (Applied Biosystems). The real‐time PCR machine (Quantabio) was programmed as follows: 95°C for 10 min, 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. Upon completion of the run, data analysis was performed (delta–delta C_t method). Genes of interest were normalized to 18S. Fold changes relative to the controls are provided. Forward and reverse primers are given in Table 1.