Reliance select cdna synthesis kit

The total RNA was isolated from the treated cardiomyocytes using the Trizol reagents (Thermo, Massachusetts, USA). The extracted RNA was treated with DNase I (Thermo, Massachusetts, USA) for 10 min to remove contaminated cDNA. The quality and concentration of RNA were validated using a Nanodrop 2000 spectrophotometer (Thermo, Massachusetts, USA). The wavelength absorption ratio (260/280 nm) was between 1.8 and 2.0 for all preparations. A total 1 µg of RNA was transcribed into cDNA utilizing the Reliance Select cDNA Synthesis Kit (Bio-Rad, California, USA). In the present study, the PCR reaction was conducted utilizing the SYBR Master Mix kit with a 25-μL reaction system and the StepOne-Plus system (Takara, Tokyo, Japan) according to the following procedure: denaturing at 95 °C for 30 s, annealing at 60 °C for 1 min and extending at 95 °C for 5 s for 40 cycles, and 72 °C 10 min, 1 cycle. Finally, the 2^−ΔΔCt method was used to determine the relative expression level of target genes with β-actin used for normalization. The following primers were used in this study: p21, (F: 5′ -TGTTCCACACAGGAGCAAAG-3′, R: 5′-AACACGCTCCCAGACGTAGT-3′), β-actin (F: 5′-CTGCCCTGGCTCCTAGCAC-3′, R: 5′- CGGACGCAGCTCAGTAACAGTCCG-3′).

Total RNA from day 12 lung tissue (40 mg) previously immersed in liquid nitrogen and pulverized with a mortar pestle on ice was extracted using the Aurum Total RNA Mini-Kit (Bio-Rad Laboratories, Hercules, CA) and quantified (Nanodrop Spectrophotometer, ThermoScientific, Waltham, MA). RNA (2 μg) exhibiting A_260/280 values of 1.8–2.0 was converted to cDNA using the Reliance Select cDNA Synthesis Kit (Bio-Rad) with random primers as previously described by Arkatkar and coworkers [15 (link)]. The resulting cDNAs were further amplified using the SsoAdvanced PreAmp Supermix (Bio-Rad). Quantitative real-time PCR was carried out using cDNA and gene-specific primer pairs (GAPDH qMmuCED002749; HSP90ab1 qMmuCED000500; CCL3 qMmuCED0044190; HMGB1 qMmuCED0041193; SOCS1 qMmuCED0024846; TBX21 qMmuCID0022343; IL-4 qMmuCED0044969, and SMS qMmuCED0040754 (Bio-Rad)) dissolved in SsoAdvanced Universal SYBR Green Supermix (Bio-Rad), and a CFX 96 instrument was used (Bio-Rad). All mRNA expression levels were normalized to housekeeping genes GAPDH and HSP90 and reported as expression relative to that of WT naïve mouse expression using the comparative cycle threshold method [16 (link)]. Quantification of gene expression was achieved using the CFX Maestro software (Bio-Rad), and results were reported as fold-change differences as previously described [14 (link), 15 (link)].