Qsybr green pcr kit

Reagents for this study included: TRIzol reagent (Thermo Fisher Scientific, USA), citrate buffer (pH=6.0) (Wuhan Boshide Corporation, China), qSYBR Green PCR kit (Shanghai GenePharma, Shanghai, China), reverse reaction kit (Promega, USA), 10×RT buffer (Dalian Bao Biotech Company, China), DEPC water (Sigma-Aldrich, USA), MMLV reverse transcriptase (Dalian Bao Biotech Company, China), 2.5 mM dNTP mixture (Nanjing Kaiji Biotech., China), 10× PCR buffer (Promega, USA), fetal bovine serum (Thermo Fisher Scientific, USA), trypsin/EDTA (Thermo Fisher Scientific, USA), Transwell chamber (Merck, USA), Lipofectamine TM 2000 (Thermo Fisher Scientific, USA), CCK-8 (Tongren Chemical Research Institute, Japan), Annexin V-FITC Apoptosis Detection Kit (Promega, USA), and RPMI-1640 medium (Life Technologies, Gaithersburg, MD).

Colonic cancer and normal colon epithelial cell lines were collected, total RNA was extracted by the TRIzol method (TRIzol reagent, Thermo Fisher Scientific, USA;qSYBR Green PCR kit, Shanghai GenePharma, Shanghai, China), RNA concentration was determined with an ultraviolet spectrophotometer, and purity was determined. Total RNA reverse transcription was synthesized in cDNA using a reverse transcription kit. The reaction conditions were 25 °C, 5 min, 42 °C, 30 min, 85 °C, 5 min and 4 °C. QPCR was amplified and detected by fluorescence quantitative PCR. QPCR reaction conditions were as follows: pre-denaturation at 95 °C for 5 min, 95 °C for 5 s, 60 °C for 30 s, 72 °C for 20 s, and 65 °C for 5 s, for a total of 40 cycles. The relative mRNA expression level of S1PR5 was calculated by 2-ΔΔCt.