Primescript rt master mix synthesis kit

According to the reference sequence of RUNX1 (XM 021068413.1), the coding sequences (CDS) were amplified and used to construct the vectors for overexpression. The primers used to amplify RUNX1 are presented in Table S1. The RUNX1 CDS fragment was ligated into the pcDNA3.1 (+) vector to obtain pcDNA3.1-RUNX1. The siRNAs of RUNX1 were synthesized by the BioRio company (Guangzhou, China). The RNAi sequences are shown in Table S1. The overexpression vectors and RNAi sequences were transfected into pGCs in a 6-well cell culture plate. The amounts of the recombinant plasmids used were 500, 750, 1000, and 1500 ng. The concentrations of the siRUNX1 fragments were 50, 75, 100, and 150 nM. The transfection groups of the pcDNA3.1 plasmids and siRNA negative sequences (siNC) were the controls of pcDNA3.1-RUNX1 and siRUNX1. Each group featured three replications. Lastly, qPCR was used to measure gene overexpression and interference. The total RNA was reverse transcribed using a PrimeScript RT Master Mix Synthesis Kit (Takara, Japan). The gene expression was quantitated by the measuring dye included in the Maxima SYBR Green qPCR Master Mix (2×) (Thermo Fisher, Waltham, CF, USA) for qPCR, and the relative expression was calculated using the 2^−ΔΔct method.

When cells covered 80% of one well, pcDNA3.1-FGFR1, pcDNA3.1-p65, pcDNA3.1-Basic, si-p65, si-FGFR1, and the negative siRNA control were transfected into the cells for 48 h. At least three wells per group were collected for extraction of total RNA. The total RNA was extracted using TRIzol reagent (TaKaRa, Tokyo, Japan) and then reverse-transcribed using a PrimeScript RT Master Mix Synthesis Kit (TaKaRa, Tokyo, Japan) for mRNAs. The relative expression levels of mRNAs were quantified using Maxima SYBR Green qRT-PCR Master Mix (2×) (Thermo Scientific, Waltham, CF, UAS) in a LightCycler Real-Time PCR system (96 system, Roche Diagnostics Ltd., Basel, Switzerland). The expression level of GAPDH mRNAs was used as endogenous controls, and the fold changes were calculated using the 2^−ΔΔct method. The primer sequences are listed in Table 1.