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′).