Trizol reagent rnaiso plus

Total RNA was extracted from cells or mouse tissues using Trizol reagent RNAiso Plus (TaKaRa, Japan) following the phenol–chloroform extraction method. Purified total RNA was used to obtain cDNA using PrimeScript RT Master Mix (Takara, Japan) following this method: 37°C for 30 min, and 85°C for 5 s. The gene expression was analyzed with the CFX Connected Real-Time PCR Detection System (BioRad) with SYBR Ex Taq Premixes (Takara, Japan). Gene expression levels were normalized to actin.

Cells were lysed in Trizol reagent RNAiso Plus (Takara, Japan) and the total RNA was isolated by standard protocol and then transcribed into cDNA using 5× Primescript^® RT Master Mix (Takara, Japan) according to the manufacturer’s instructions. Gene expression was determined by qRT-PCR analysis by using 2× SYBR^® Green Mix (Takara, Japan) on a Bio-Rad detection system (Bio-Rad). The primers used in this study are listed below. hEGFR-F: 5′- GGCACTTTTGAAGATCATTTTCTC-3′, hEGFR-R: 5′-CTGTGTTGAGGGCAATGAG-3′; hXIAP-F: 5′-AGTGGTAGTCCTGTTTCAGCATCA-3′, hXIAP-R: 5′-CCGCACGGTATCTCCTTCA-3′; hBCL-2-F: 5′-CATGCTGGGGCCGTACAG-3′, hBCL-2-R: 5′-GAACCGGCACCTGCACAC-3′; hBCL-XL-F:5′-TGCATTGTTCCCATAGAGTTCCA-3′, hBCL-XL-R: 5′-CCTGAATGACCACCTAGAGCCTT-3′; hSurvivin-F:5′-TGCCTGGCAGCCCTTTC-3′, hSurvivin-R: 5′-CCTCCAAGAAGGGCCAGTTC-3′.

For replicative senescence, total RNA was extracted from different passages of HUVEC cells (P6, P20 and P30) by TRIzol reagent RNAiso Plus (Takara). Library construction, sequencing and bioinformatic analysis were done by Origingene Bio-pharm Technology Co. Ltd. (Shanghai). The P value is calculated by edgeR (v3.24). FDR (False Discovery Rate) is obtained by multiple testing and adjusting P value. The differentially expressed genes (DEGs) were defined as P < 0.05 and log₂(FC) ≥0.75 or log₂(FC) ≤−0.75. DEGs were used for KEGG pathway analysis and KOBAS software was used to test the statistical enrichment of DEGs in KEGG pathways.
For RT-qPCR, RNA is extracted from cells with TRIzol reagent RNAiso Plus (Takara). Purified RNA was digested with DNase (Sigma) and reversed transcribed into cDNA using Reverse Transcriptase Kit (M-MLV) (ZOMANBIO). The cDNA was quantitated by qPCR with SYBR Green premix (Yeasen) using primers listed in Supplementary Table 1. The mRNA level of the gene of interest was normalized to that of ACTIN.

Total RNA was isolated using Trizol reagent RNAisoPlus (Takara, Dalian, China) and reversely transcribed into cDNA using Primescript RT Master Mix (Takara), after which, expression of target gene was evaluated by qRT-PCR using SYBR Green Mix (Takara) according to the manufacturer′s instructions. The primes used were listed as follow: linc00261: 5′-GTCAGAAGGAAAGGCCGTGA-3′ (forward), 5′-TGAGCCGAGATGAACAGGTG-3′ (reverse); Nanog: 5′-TGAACCTCAGCTACAAACAG-3′ (forward), 5′-TGGTGGTAGGAAGAGTAAAG-3′ (reverse); SOX2: 5′-ACGCTCATGAAGAAGGATAAGT-3′ (forward), 5′-GAGCTGGTCATGGAGTTGTAC-3′ (reverse); OCT4: 5′-AGGTGGTCCGAGTGTGGTTC-3′ (forward), 5′-GAGGAGTACAGTGCAGTGAAGTG-3′ (reverse); Slug: 5′-CTGTGACAAGGAATATGTGAGC-3′ (forward), 5′-CTAATGTGTCCTTGAAGCAACC-3′ (reverse); Snail: 5′-CTTCCAGCAGCCCTACGAC-3′ (forward), 5′-CGGTGGGGTTGAGGATCT-3′ (reverse); ZEB1: 5′-AGCAGTGAAAGAGAAGGGAATGC-3′ (forward), 5′-GGTCCTCCTCAGGTGCCTCAG-3′ (reverse); 18SrRNA, 5′-GTAACCCGTTGAACCCCATT-3′ (forward), 5′-CCATCCAATCGGTAGTAGCG-3′ (reverse). 18S rRNA was used as internal control, and 2^−ΔΔCT method was applied to analyze expression of target genes.