Easy blue total rna extraction kit

Total RNA was extracted using an Easy-Blue™ Total RNA extraction kit according to the manufacturer's instructions and reverse transcription of RNA to cDNA was performed using an ABI cDNA synthesis kit (Applied Biosystems, Foster City, CA, USA) (conditions: 37°C for 1 h, followed by 95°C for 5 min). TaqMan quantitative RT-PCR with an ABI StepOne Plus detection system was performed according to the manufacturer's instructions (Applied Biosystems; Thermo Fisher Scientific, Inc.). For each sample, triplicate test reactions and a control reaction without reverse transcriptase were analyzed for expression of the gene of interest and to control for variations in the reactions. All qPCR data were normalized to levels against the housekeeping gene hypoxanthine guanine phosphoribosyltransferase (HPRT). Forward, reverse, and probe oligonucleotide primers for multiplex real-time TaqMan PCR were purchased from ABI (Applied Biosystems; Thermo Fisher Scientific, Inc.). Cycling conditions were 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec. The data were analyzed using StepOne™ software (version 2.3; Applied Biosystems; Thermo Fisher Scientific, Inc.). The 2^−ΔΔCq method was used to determine the relative mRNA expression level (20 (link)).