Gdna removal kit

An RNA isolation system (Sigma-Aldrich, St. Louis, MO, USA) was used to extract total RNA and a Prime Script RT reagent kit with a gDNA removal kit (Takara Bio, Otsu, Japan) was used in obtaining cDNA. A 20 µL total reaction system containing 10 uL of SYBR (Takara Bio, Otsu, Japan), 0.5 µM forward primer, 0.5 µM reverse primer, 5 ng/µL cDNA, and DEPC water was used. A qRT-PCR machine was set at 95 °C for 2 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. Data were analyzed using comparative 2^−△△Ct with normalization to the Gapdh housekeeping gene. The primers were synthesized in Kumei, Changchun, China (Table 1).

The total RNA from GCs was extracted using TRIzol reagent (Invitrogen, Foster City, CA, USA) following the manufacturer’s protocol. cDNA was synthesized using a Prime Script RT Reagent Kit with a gDNA Removal Kit (Takara Bio, Otsu, Japan). The total volume for the RT-qPCR reaction was 20 μL, comprising 10 μL of SYBR Green (Takara Bio, Otsu, Japan), 0.5 μL of each forward and reverse primer, 4 μL of cDNA, and 5 μL of DEPC-treated water. The RT-qPCR cycling conditions were as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 30 s, and annealing at 60 °C for 1 min. The data were analysed using the comparative 2^−ΔΔCt method, with normalization to the GAPDH housekeeping gene. Primers were custom synthesized by Comate Bioscience Co., Ltd. (Changchun, China) (Table 1).