Microrna reverse transcription kit

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.

Total RNA content was extracted from the tissues and cells using the TRIzol agent (16096020, Thermo Fisher Scientific). The cDNA of miRNA was synthesized using the miRNA First Strand cDNA Synthesis kit (Tailing Reaction) (B532451, Sangon Biotech), while the mRNA was obtained using the BeyoRT^TM II First Strand cDNA Synthesis kit (RNase H-) (D7168L, Beyotime, Shanghai, China). Subsequently, RT-qPCR was performed using the AceQ^® Universal SYBR qPCR Master Mix kit (Q511-02, Vazyme Biotech, Nanjing, China) on a CFX96^TM Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, United States). U6 was regarded as the internal control for miR-324-3p, while glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal control for lncRNA 74.1 and NRG1. The primer of U6 was obtained using the microRNA reverse transcription kit, and the primer sequences of lncRNA 74.1, miR-324-3p, NRG1, and GAPDH were provided by Sangon Biotech (Supplementary Table 1). Fold changes were calculated based on the 2^–ΔΔCt method.