Cells were cultured for 48h in the original IVF plate, washed 3 times with DMEM/Ham's F12 (1:1) supplemented with penicillin, streptomycin and amphotericin and resuspended in the same medium supplemented with 10% Fetal calf serum. Cells were incubated for 48h at 37°C with 5% CO2, medium was replaced after 24h.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using Trizol (Molecular Research) according to the manufacturer's instructions
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the Small Sample Target Labeling Assay v.2' protocol from 50 ng total RNA from Affymetrix, (Santa Clara, CA)
Hybridization protocol
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on Affymetrix GeneChip Human Genome U133 Plus 2.0 Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
Scan protocol
GeneChips were scanned using the Agilent GeneArray Scanner (Affymetrix)
Description
All the participants included in the study were women undergoing In-Vitro Fertilization with intracytoplasmatic sperm injection (IVF-ICSI) at the IVF unit. The IVF-ICSI cycles included in the study were conducted according to the long mid luteal GnRH agonist (Diphereline 3.75 mg PharmaBiotech, France) protocol. Controlled ovarian hyperstimulation was conducted with either recombinant follicular stimulating hormone (rFSH- Follitropin Alfa, Serono, UK) or human menopausal gonadotropin (hMG - Menotropin, Ferring, Germany). Following oocyte retrieval, cumulus cells were stripped from the oocyte, in preparation for the ICSI process, with a micropipette.
Data processing
Data was preprocessed using Robust Multiarray Average (RMA) with default parameters, through the Affy package of bioconductor.
