DRP007984
miRNA secreted from porcine oocyte and granulosa cells complexes
Quality of oocytes is important for successful pregnancy. Oocytes develop under well-orchestrated interactions with granulosa cells and follicular fluid. Follicular fluid is a solo environment for oocyte and granulosa cell development and the quality of follicular fluid is known to affect oocyte development. Follicular fluid contains many biological factors including exosome. Exosome contains DNA, mRNA and miRNA, and miRNA in exosome of follicular fluid support oocyte development and granulosa cells proliferation. miRNA secreted from granulosa cells contribute to the miRNA pool in follicular fluid. This data is miRNA profile found in the medium of oocyte and granulosa cells (OGCs). Oocyte and granulosa cells complexes (OGCs) were collected from early antral follicles (0.5-0.7mm in diameter) of porcine ovaries and were cultured 1in 96 well plate for 14 days. Bottom of the each wells were paved with poly acryl amid gel gels (PAG). PAG was prepared using N,N-methylenebisacrylamide (0.3%), water, acrylamide (10%), ammonium peroxodisulfate and N,N,N',N'-tetramethylethylenediamine. The PAG sheets were washed and incubated in PBS overnight. The PAGs were hollowed out to fit the bottom of the wells of a 96-well plate (Dickinson, Franklin Lakes, NJ, USA) and set at the bottom of 96 well plates. The medium used for in vitro growth of OGCs was alpha MEM (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 2% polyvinylpyrrolidone (Sigma-Aldrich), 26 mM NaHCO3, 2 mM hypoxanthine, 10 mM taurine, 0.3% BSA (Fraction-V), 1 Âµg/mL 17 betaÂ·Estradiol (E2), 0.1 mAU/mL FSH (Kyoritsu Seiyaku Corporation, Tokyo, Japan) and Insulin-Transferrin-Selenium (final concentration Ã?100 (Gibco BRL, Paisley, UK). During the culture period half of the medium was changed with fresh medium every 4 days. The culture wells were equilibrated overnight in IVG medium. Prior to experimentation, the medium was replaced with fresh IVG medium. At the end of the culture period, medium was collected and exosome in the medium were extracted using extraction kit (SBI, RA800TC). RNA quality and concentration were examined using a Bioanalyzer (Agilent technologies, Palo Alto, CA, USA) and the RNA was used for cDNA library construction using SMARTerÂ® smRNA-Seq Kit for Illumina. Sequencing was conducted via Nextseq (Illumina) to sequence 75 bp (single read) and raw reads, which were trimmed by removing 3-end poly A adapters and 5-end 3 oligos. In addition, sequence data less than 15bp and over 50 were discarded. Data quality was re-checked, following trimming. Small RNA reads were mapped to miRbase 22.1 (http://www.mirbase.org/) to identify known pig miRNAs using CLC software (Qiagen)