Hief unicon qpcr sybr green master mix

Total RNA was isolated from intestine, kidney, liver, spleen and testis tissues, respectively, using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.). RNA quantity and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). For cDNA synthesis, 2,000 ng RNA was reverse transcribed using Hifair^® III 1st Strand cDNA Synthesis SuperMix for qPCR (Shanghai Yeasen Biotechnology Co., Ltd.). qPCR was conducted on a StepOne Real-Time PCR System (ABI StepOnePlus, Applied Biosystems; Thermo Fisher Scientific, Inc.) using Hief UNICON^® qPCR SYBR Green Master Mix (Shanghai Yeasen Biotechnology Co., Ltd.). The following thermocycling conditions were used for the qPCR: Initial denaturation at 95°C for 1 min; followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. The primers were synthesized by BioSune Biotechnology. The following primers were used: Ptgs2 forward 5′-ATACGCTGAGTGTGGTTTGC-3′ and reverse 5′-CTCACTAAACCATCCAATCGG-3′; GAPDH forward 5′-TGCGACTTCAACAGCAACTC-3′ and reverse 5′-GCCTCTCTTGCTCAGTGTCC-3′. Ptgs2 fold changes were calculated after normalizing the change in expression of GAPDH using 2^−ΔΔCq method (15 (link)). The experiments were repeated in triplicate.