Rna isolater

Total RNA was extracted from the hippocampus tissues homogenized in RNA isolater (Vazyme Biotech Co.,Ltd, Nanjing, China). Using a spectrophotometer (DU800, Beckman Coulter Inc., Brea, CA, USA), the total RNA concentration was confirmed at 260 nm and 280 nm. Then, 1 μg of purified RNA was reverse-transcripted for RT-PCR to generate cDNA with HiScript II Q RT SuperMix for qPCR (+g DNA wiper) (Vazyme Biotech Co.,Ltd, Nanjing, China). qPCR was performed using the ChamQ SYBR qPCR Master Mix (Vazyme Biotech Co.,Ltd, Nanjing, China) and determined on a real-time PCR detection system (Roche, Switzerland). The relative mRNA expression level was determined with the 2-ΔΔCt method with β-actin as the internal reference control. Primer sequences were shown in Supplementary Table 1.

Yeast cells were grown to OD₆₀₀ of ~ 0.6 in 50 mL cultures. Cells were collected by centrifugation at 3000 rpm for 10 min and washed twice with 10 mL sterile water. Cells were resuspended in 0.4 mL RNA isolater (Vazyme Biotech Co.), snap-frozen in liquid nitrogen, and stored at −80°C freezer. Total RNAs were extracted using RNA isolater, and purified via chloroform extraction method. The purity and yield of RNAs were examined by measuring the A₂₆₀/A₂₈₀ ratio using NanoDrop One Spectrometer (Thermo Scientific). RNA integrity was examined by agarose gel electrophoresis. An aliquot of 1 μg total RNA from each sample was subjected to reverse transcription using the HiScript II Q RT SuperMix for qPCR (Vazyme Biotech Co.) according to the manufacturer's protocol. Quantitative PCR (qPCR) experiments were carried out as the manufacturer's protocol suggested. The Cq values of all amplification curves were less than 28 and the melt curves of each primer set only contain a single peak. Three technical replicates data were analyzed using Bio-Rad CFX Manager software (version 3.1) with normalized mode (ΔΔCq). The unpaired two-tailed Student's T-test was used to examine the statistical significance of difference in two samples.

Total RNA was extracted from Hep3B cells which transfected with shNC and shIGF2BP3 lentivirus, according to the RNA Isolater (Vazyme, China). The concentration and purity of total RNA were measured. Through SureScript First-strand cDNA synthesis kit (GeneCopoeia, USA), total RNA was used to obtain cDNA by reverse transcription reaction. The RT-PCR assays were carried out by a protocol from Power SYBR Green (Takara, Hangzhou, Zhejiang, China). The relative expressions of genes were calculated and normalized using the 2^−ΔΔCt methods relative to GAPDH. Specific primer sequences were as follows: IGF2BP3: 5′-ACGAAATATCCCGCCTCATTTAC-3′ (forward), 5′-GCAGTTTCCGAGTCAGTGTTCA-3′ (reverse); GAPDH: 5′-CTGGGCTACACTGAGCACC-3′ (forward), 5′-AAGTGGTCGTTGAGGGCAATG-3′ (reverse).

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.