Expression Analysis of IR-lncRNAs in CRC

The UALCAN database was employed to verify the expression level of the six IR-lncRNAs in CRC tumor and non-tumor samples (Chandrashekar et al., 2017 (link)). Furthermore, 36 matched tumor and non-tumor tissue specimens were collected from Ruijin Hospital, Shanghai Jiao Tong University School of Medicine after approved by the local ethics committee of Ruijin Hospital (No. 2020-384). The patients did not receive any form of treatment before specimen collection. The total RNA of tissue specimens was extracted with RNA isolater (Vazyme, China), and then, one μ g of total RNA was reverse transcribed into cDNA with Hiscript III RT Supermix and gDNA wiper (Vazyme, China). ChamQ universal SYBR qPCR Master Mix (Vazyme, China) was used for real-time fluorescence quantitative PCR (qRT-PCR) analysis, and the expression level of GAPDH served as an internal control. The cycle scheme is 95°C for 5 min, 40 cycles at 95°C for 15 s, 60°C for 60 s, 72°C for 5 min. All primers were synthesized by Tsingke (Beijing, Shanghai) company and primer sequences are presented in Table 2. The comparative Ct (2^−ΔΔCt) method was adopted to calculate the relative expression level of the six IR-lncRNAs.