Evaluation of the in vitro effects of irisin and leptin on human ovarian granulosa cells – PCR array data (female infertility panel)
Description
Human ovarian granulosa cells were purified from follicular fluid samples obtained from patients undergoing in vitro fertilization procedure by using Percoll PLUS reagent (GE Healthcare, Cat. # 17-5445-02). Cells were grown in DMEM/F12 (50:50) medium (Corning, Cat. # 10-092-CM) supplemented with 10% FBS (VWR, Cat. # 89510-186) and antibiotic/antimycotic mixture (MP Biomedicals, Cat. # 1674049). For experiment, 0.3 x 10(6) cells were seeded in 6-well plates and the following day the cell culture medium was replaced with serum-free medium supplemented with 500 ng/ml of irisin (Enzo, Cat. # ADI-908-307-0010) or 50 ng/ml of leptin (BioVision, Cat. # 4366-02) for 24 hours. After completing the experiment, cells were washed two times with PBS (Corning, Cat. # 46-013-CM) and total RNA was extracted using TRIzol reagent (Ambion, 15596018). RNA was quantified using NanoDrop One spectrophotometer (Thermo Fisher Scientific) and samples were normalized to 1 mg/ml concentration, then converted to cDNA using qScript cDNA SuperMix (Quantabio, Cat. # 95048). PCR array was performed using RT2 Profiler PCR Array – Human Female Infertility (Qiagen, Cat. # 330231, GeneGlobe ID PAHS-164Z) and QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific) following manufacturer’s protocol. PCR array data were calculated using GeneGlobe Data Analysis Center (https://geneglobe.qiagen.com/us/analyze).