Rna quick purification kit

Total RNA was extracted from the mBMSCs using the RNA-Quick Purification Kit (Vazyme, China) and the cDNA was amplified using the HiScript II Q RT SuperMix for qPCR (Vazyme) according to the manufacturer’s instructions. The qPCR was performed with SYBR Green PCR Master Mix (Vazyme) on using ABI steponeplus real-time PCR system (Applied Biosystems, USA). The level of expression was standardized to GAPDH, and the relative expression level was evaluated using the 2^−ΔΔCT approach. The primers used in this experiment were synthesized by GENEbay (China) with the following sequence: GAPDH (Forward: 5′-TCATGGGTGTGAACC ATGAGAA-3′, Reverse: 5′-GGCATGGACTGT GGTCATG AG-3′), COL II (Forward: 5′-CACACTGGTAAGTGGGGCAAGACCG-3′, Reverse: 5′-GGATTGTGTTGTTTC AGGGTTCGGG-3′), ACAN (Forward: 5′-CCTGCTACTTCATCGACCCC-3′, Reverse: 5′-AGATGCTGTTGACTCGAACCT-3′), and SOX9 (Forward: 5′-GGTGC T CAAGGGCTACGACT-3′, Reverse: 5′-GGGTGGTCTTTCTTGTGCTG-3′).

Cells (2.0 × 10⁵) were seeded in six-well plates and then IGF2BP2 shRNA or RUNX2 siRNAs were transfected into cells. RNA-Quick Purification Kit (Vazyme, Nanjing, China) was used for total RNA isolation from cells. 1 µg total RNA was converted to cDNA for RT-qPCR using HiScript II Q RT SuperMix for RT-qPCR (Vazyme, Nanjing, China). An Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems) using AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China) was used for real-time quantitative RT-qPCR analysis. To standardize the input cDNA, β-actin was run in parallel as reference. The primer sequences are provided in Supplementary Table S1.

To further confirm the expressions of the four immune-related lncRNAs in cells and tissues, human normal lung epithelial cell (B2B) and lung adenocarcinoma cell lines (A549, H1299, H1975) were obtained from the laboratory and cultured in DMEM medium containing 10% FBS with 1% penicillin and streptomycin in a humidified incubator. The total RNA of various cell lines was extracted using RNA-Quick purification kit according to manufacturer’s instructions (Vazyme, RN001). Total RNA were reversed into cDNA using PrimeScript™ RT reagent kit with gDNA Eraser (Takara, RR047A). Next, we performed quantitative PCR to determine the relative expression levels of the four lncRNAs (Takara, RB820A). The tissues’ expressions and survival curves of the four lncRNAs were acquired from the GEPIA database (http://gepia.cancer-pku.cn/). The expression of β-actin was used as an endogenous control. All samples were analyzed using comparative 2^−ΔΔC method. All primers’ sequences used in PCR were shown in Table S1.