Fastpure dna isolation

TRIzol reagent (Takara, Japan) was utilized for isolating complete RNA from CRC tissues, normal tissues along with cell lines. gDNA (genomic DNA) was extracted using FastPure DNA Isolation (Vazyme, China). Nanodrop 2000 was used to examine the RNA samples’ purity as well as concentration (Thermo Fisher Scientific, USA). mRNA and circRNA were reversely transcribed utilizing the PrimeScript RT master mix (Takara, Japan) and designed primers, with glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) as an internal control. Reverse transcriptions for miRNA were carried out utilizing Bulge‐Loop™ miRNA quantitative real‐time polymerase reaction (qRT‐PCR) starter kit (RIBOBIO, China) and unique stem‐loop primers, with U6 as an internal control. cDNA was amplified with the ABI Prism 7500 sequence detection system and TB Green Premix Ex Taq II (Takara, Japan) (Applied Biosystems, USA). These procedures were carried out for each sample in triplicate. The 2^−ΔΔCT technique was utilized for quantifying the circRNA, miRNA, and mRNA expression levels with the internal regulation. The back‐splice junction of circRNA was detected using divergent primers, while linear mRNA was detected using convergent primers. The primers are listed in Table S1 the Supporting Information.

Total RNA was isolated from tumor samples and cell lines using TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA) and genomic DNA (gDNA) were isolated using FastPure DNA Isolation (Vazyme, Nanjing, China). For circRNA and mRNA, reverse transcriptions were performed using the PrimeScript RT Master Mix (Takara, Shiga, Japan) with random primers. For miRNA, reverse transcriptions were performed using the PrimeScript RT Reagent Kit (Takara) with specific stem-loop primers and cDNA amplification performed using TB Green Premix Ex Taq II (Takara). GAPDH and U6 were used as internal control and each sample was repeated in triplicate. The relative fold-change in expression with respect to a control sample was calculated by the 2-ΔΔCt method. Divergent primers were used to detect backsplice junction of circRNA and convergent primers were used to detect linear mRNA. The primers for miR-BART19-3p and U6 small nuclear RNA were obtained from RiboBio Company (Guangzhou, China). The primers were listed in Table S1.