Sybr green qpcr master mix

The ovarian cancer cell line (SKOV3) and human normal ovarian epithelial cell line (IOSE80) were purchased from China Center for Type Culture Collection (Wuhan, China). Fetal bovine serum (FBS) and Roswell Park Memorial Institute (RPMI) 1640 media were obtained from Gibco (Shanghai, China). Total RNA extraction kit, microRNA Reverse Transcription Kit, and 2× SYBR Green qPCR master mix were supplied by Sangon Biotech Co., Ltd. (Shanghai, China). RNA-related operations required the involvement of Rnase-free water.

For the isolation of total RNA, 50–100 mg of freezing sample was ground with liquid nitrogen in a pre-treated RNase free mortar. Total RNA was extracted from the ground sample with the classical Trizol method as we described in a previous article (Yang et al., 2019 (link)). The obtained total RNA was dissolved in 30–50 μl of RNase free water. The concentration and purity of the RNA were determined using a nanodrop UV spectrophotometer. In general, RNA was quantified for the following cDNA synthesis when the value of A260/A280 was above 1.90. cDNA was produced in an ABI Veriti 96 Well Thermal Cycler (Waltham, MA, United States) using FastQuant RT Kit from Tiangen Biotech Co., LTD. (Beijing, China). Real-time PCR was performed in an ABI QuantStudio3 PCR System (Waltham, MA, United States) using SYBR Green qPCR Master Mix and gene specific primers synthesized by Sangon Biotech (Shanghai, China). The program was set as following: initial denaturation at 95°C for 10 min followed by 40 cycles of 95°C for 15 s, 58°C for 30 s and 68°C for 60 s. The primers used in this study were listed in Table 1. The relative expression of target mRNA was calculated by normalizing target mRNA C_ts to the housekeeping gene GAPDH (method of 2^-DDCt) (Yang et al., 2019 (link); Yin et al., 2019 (link)). Undetectable of the target mRNA was defined as the C_t value greater than 30 cycles.