Rna trizol reagent

For evaluating the expression levels of three signature regulators and hub genes, we extracted the total RNA from clinical GEA samples by using RNA trizol reagent (CWBIO). According to the instructions of the manufacturer, cDNA synthesis was carrying out by using the reverse transcription kit (CWBIO). The qRT-PCR analysis was conducted on the LightCycler 480 Real-Time PCR System. The PCR mixtures were preheated for 5 min at 95°C, followed by 45 cycles of 95°C for 10 s, and 60°C for 45 s, and the final dissolution curve analysis was performed according to manufacturer's instruction. Related gene expression levels were calculated using the 2^−△△CT method and the related GAPDH mRNA expression was used as an endogenous control. Primer sequences are presented in Supplementary Table S2.

We extracted total RNA from the cells using RNA TRIzol reagent (CWBIO) to assess the expression levels of the five prognostic MRGs. cDNA synthesis was performed using a reverse transcription kit (CWBIO) according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was conducted on a LightCycler 480 Real-Time PCR system. The relative mRNA expression level was calculated by using the 2^−ΔΔCt approach and standardized to β-actin. Supplementary Table S2 showed the primer sequences.