Beyort 3 cdna synthesis regent

The mRNA in the clinical tissues and cultured cells were extracted. Briefly, the tissue and cell samples were mixed with Trizol (R21086, OKA, Beijing, China) and chloroform (A23482, OKA) for 30-min centrifugation (14,000 × g) to collect the supernatant. Then, the supernatant was mixed with isopropanol (E15794, OKA) and further centrifuged (14,000 × g) for 15 min. After the mRNA was collected from the precipitations, it was synthesized into cDNA using BeyoRT III cDNA synthesis regent (D7178L, Beyotime). Finally, the cDNA amplification was performed by mixing the cDNA with the Supermix (AQ601-01, TransGen, Beijing, China) and primers of target genes in the QuantStudio6 system (Applied Biosystems, CA, USA). After the amplifying reaction, the target gene expressions were quantified using the 2^−ΔΔCt method. The primers used in the experiments were as follows: MYC-F: 5ʹ-GGCTCCTGGCAAAAGGTCA-3ʹ, MYC-R: 5ʹ-CTGCGTAGTTGTGCTGATGT-3ʹ; NBS1-F: 5ʹ-GACTGGCGTTGAGTACGTTGT-3ʹ, NBS1-R: 5ʹ-TGATTTCGGCTGATCGACTGA-3ʹ; MRE11-F: 5ʹ-TCCGTGAGGCTATGACCAGG-3ʹ, MRE11-R: 5ʹ-TTGGTTGCTGCTGAGATGCTAT-3ʹ; β-actin-F: 5ʹ-CCACGAAACTACCTTCAACTCC-3ʹ, β-actin-R: 5ʹ-GTGATCTCCTTCTGCATCCTGT-3ʹ.