RNA Extraction, Reverse Transcription and Quantitative Real-time PCR
Isolated ovarian GCs were lysed by Trizol reagent (Thermo Fisher Scientific, Inc.) for 30min to extract total RNA according to the guidance. The concentration and purity of the total RNA was determined by UV spectrophotometry. The ratio of the optical density at 260/280 nm was between 1.8 to 2.1. Total RNA was reverse transcribed into cDNA using Prime Script RT Reagent Kit (TaKaRa, Japan) for real-time quantitative PCR(RT-qPCR) carried out by TB Green™ Premix Ex Taq™ II (TaKaRa, Japan) and performed on Roche Light Cycle 480 (Hoffman-La Roche, Switzerland). ANGPTL4 mRNA expression levels were normalized against the corresponding levels of GAPDH mRNA, which were served as an internal control and the 2-∆∆Ct method13 was applied to determine ANGPTL4 expression level. Primer sequences were as follows: ANGPTL4 (forward primer, 5’-TCCTGGACCACAAGCACCTAGAC-3’; reverse primer, 3’-CGGTTGAAGTCCACTGAGCCATC-5’), GAPDH (forward primer, 5′-GCACCGTCAAGGCTGAGAAC-3′; reverse primer, 3′-TGGTGAAGACGCCAGTGGA-5′).