Hiscript q select rt supermix

The EPC cells were divided into three groups: control_DMSO, TET, and DHA. All samples were harvested 24 and 48 h after incubation, then extracted using TriQuick Reagent (Solarbio, China). The cDNA was synthesized using HiScript Q Select RT SuperMix (Vazyme, China) and qPCR was performed on an ABI QuantStudio 3 Real-Time PCR System (Applied Biosystems, USA) using AugeGreeen qPCR Master Mix (US Everbright Inc., China). The primers used for qPCR analysis are listed in Supplementary Table S1. The relative expression levels of genes were analyzed using the 2^−△△CT method, with β-actin as the reference.

Fresh tissue samples were immediately frozen in liquid nitrogen after surgical resection. Total RNA from tissues and cells was extracted using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized using the HiScript Q Select RT SuperMix for quantitative PCR and random hexamers from Vazyme Biotech (Nanjing, China). Quantitative real-time PCR was carried out with the SYBR Green PCR Master Mix (Vazyme Biotech). PCR primers are as follows: MCM10 forward, 5′-CACAGAAATGAACAAGAA-3′ and MCM10 reverse, 5′-AATAAGAACAAGGACACA-3′; GAPDH forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and GAPDH reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′. GAPDH was used as a loading control. The relative expression of MCM10 was calculated using the 2^−ΔΔCt method.13 (link)

Total RNA was isolated from cell cultures or from snap-frozen tissues from GC patients using RNAiso Plus (Takara Bio, Kusatsu, Japan) according to the manufacturer's instructions, reverse transcribed with HiScript Q Select RT SuperMix (R132-01; Vazyme, Jiangsu, China) according to the manufacturer's protocol, and subjected to real-time reverse transcription (RT)-PCR using the 2^−ΔΔCq method (21 (link)). The thermocycling conditions were as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 10 sec, 60°C for 32 sec, 95°C for 15 sec, 60°C for 60 sec and 95°C for 15 sec. Each sample was determined in duplicate. All PCR products were confirmed by 2.0% agarose gel electrophoresis. The sequences for RT-PCR primers were: CPXM2 forward primer, 5′-GTGCGCGGGAAGAAATGAC-3′ and reverse primer, 5′-CCTCCCTTGAGTGATGACACC-3′. The specificity of primers was validated by sequencing. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (forward primer, 5′-GTCAAGGCTGAGAACGGGAA-3′ and reverse primer 5′-AAATGAGCCCCAGCCTTCTC-3′) served as an internal control. Experiments were repeated three times in duplicate.