Cfx 192 connect real time pcr system

A total of 14 pairs of GC samples (Additional file 1: Table S1) were collected and the relative expression of genes in the RS model was detected. The study was approved by the Ethics Committee of Renmin Hospital of Wuhan University (No. NCT03972956V1.1), and we have obtained informed consents from the patients for using GC samples (No. SAMPGICU2019-2). Total RNA was extracted using Tissue Total RNA Isolation Kit (Foregene, Wuhan, China) as per the manufacturer’s protocol and RNA (2 μg) was reverse transcribed to cDNA using the PrimeScriptTM RT reagent Kit (TaKaRa, Osaka, Japan). Quantitative PCR (qPCR) was performed using the SYBR Green qPCR Supermixes (Bio-Rad) on the CFX 192 Connect Real-Time PCR system (Bio-Rad, USA). The qPCR analysis was performed in triplicates with the designed primers (Table 1). The relative expression was normalized to GAPDH using the 2-ΔΔCT method.Table 1

List of primers

Gene	Primer sequence (5′–3′)
SLIT2	Forward: GCACCATTGAAAGAGGAGCA
	Reverse: GCTTTCCTTGGGATTGCCTG
SFRP2	Forward: ACCGAGGAAGCTCCAAAGGTA
	Reverse: GAGCCACAGCACCGATTTCT
SCRG1	Forward: CATTTCTGGGATGGGAAGGGA
	Reverse: GTGGGAAATCAGGAATGGTGTT
MFAP5	Forward: CAGCGTAAGAGGAGAGAGACAC
	Reverse: CAGCAAGAAACAGCAGCACCT
EFEMP1	Forward: TGAAATGCAGACTGGCCGAA
	Reverse: TCTACAGTTGTGCGTCCCTG
COL8A1	Forward: AAGGAGATGCCCCACTTGC
	Reverse: GGACCTTGTTCCCCTCGTAA
ABCA8	Forward: AAGAACGCAAAACAGACCGC
	Reverse: TTTGGCATCAGGGATGTGCT

Total RNA was extracted using Cell Total RNA Isolation kit (Foregene, Chengdu, China) following the manufacturer’s protocol, and RNA (1 μg) was reverse‐transcribed to cDNA using the PrimeScript RT reagent kit (TaKaRa, Osaka, Japan). Quantitative real-time PCR (qPCR) was conducted using the SYBR Green qPCR Supermixes (Bio-Rad) on the CFX 192 Connect Real-Time PCR system (Bio-Rad, U.S.A.). The qPCR analysis was performed in triplicate with the primers shown in Table 2. The relative expression levels were normalized to GAPDH using the 2^−ΔΔCT method.