Random hexamer primers

Total RNA was isolated from 100 mg of cardiac
muscles using RNX ^TM isolation reagent according to
the manufacturer’s procedure (CinaClon, Iran). Possible
DNA contamination was removed by treatment of
RNA (1 µg) with DNase I (2 U/µl) for 1 hour at 37oC
(Vivantis, Malaysia). Concentration of extracted RNA
was calculated at the wavelength of 260 nm using
NanoDrop spectrophotometer (Eppendorf, Germany). To
detect the purity of RNA, its optical density (OD) ratio at
260/280 nm was determined and samples having a ratio
>1.8 were used for cDNA synthesis. Reverse transcription
was carried out using the RocketScript RT PreMix kit
using 1 µg of RNA and random hexamer primers based
on manufacturer’s protocol (Bioneer Corporation, South
Korea). Reverse transcription was carried out at 42°C for
90 minutes followed by incubation at 70°C for 5 minutes.
cDNAs were stored at -20°C until used for real-time
polymerase chain reaction (PCR).

Total RNA was isolated from 100 mg of AT using RNX TM isolation reagent according to the manufacturer's procedure (Cina Clon, Iran). Possible DNA contamination was removed by the treatment of RNA (1 μg) with DNase I (2 U/μl) for 1 hr at 37°C (Vivantis, Malaysia). The concentration of extracted RNA was calculated at the wavelength of 260 nm using NanoDrop spectrophotometer (Eppendorf, Germany). For detecting the purity of the RNA, the optical density (OD) ratio at 260/280 nm was determined, and samples with a ratio >1.8 were used for cDNA synthesis. Reverse transcription was carried out using the Rocket Script RT PreMix kit with 1 μg of RNA and random hexamer primers based on the manufacturer's protocol (Bioneer Corporation, South Korea). Reverse transcription was carried out at 42°C for 90 min, followed by incubation at 70°C for 5 min. cDNAs were stored at −20°C to be used later for real‐time polymerase chain reaction (PCR).

Total RNA was isolated from cell cultures using the TRI reagent (MRC, Inc., Cincinnati, OH), and 2 µg of RNA was reverse-transcribed into cDNA using random hexamer primers (Bioneer, Daejeon, Korea) and Superscript III reverse transcriptase (Invitrogen, Grand Island, NY) in a thermal cycler (Eppendorf, Happauge, NY) according to the manufacturer's instructions. Quantitative RT-PCR was carried out in a total volume of 10 µL containing 5 µL of LightCycler® 480 SYBR Green I Master (Roche Diagnostics Ltd., Rotkreuz, Switzerland), 0.5 µM of each primer and 2.5 µL of 1∶10 diluted cDNA using LightCycler® 480 instrument (Roche Diagnostics Ltd). The cycling conditions were: 95°C for 5 min, followed by 45 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 10 s. All the samples were carried out in triplicates. The expression levels of each mRNA expression were normalized to the housekeeping gene GAPDH using LightCycler® 480 Software, Version 1.5 (Roche Diagnostics Ltd). Primer sequences for FOXG1, DLX2, NKX2.1, GAD1, calbindin2 (CALB2) and somatostatin (SST) have been previously described [26] (link). Other primer sequences were retrieved from PrimerBank Database http://pga.mgh.harvard.edu/primerbank/[27] (link), with the exception of SLC32A1 which was designed using ProbeFinder software Version 2.49 from Roche Applied Science. Sequences of primer sets were listed in Table S1.