7 protocols
Standard Illumina scanning protocol
The data were transformed using the variance stabilization transformation method (Lin et al. 2008) and normalized by robust spline normalization (Du et al. 2008) using R 2.15.2 and the lumi 2.8.0 package from Bioconductor.
mRNA samples were in vitro labeled using the TargetAmp 1-Round Aminoallyl-aRNA Kit with biotin (Epicentre, Madison, WI).
ID_REF = VALUE = Vst transform and rsn normalized
Standard Illumina hybridization protocol. Samples were randomly hybrized to BeadChips.
RNA was purified from follicles using the Qiagen RNeasy Micro Kit according to the manufacturer’s protocol (Qiagen, Valencia, CA). RNA quality and quantity was assessed both by NanoDrop (Thermo Scientific, Wilmington, DE) and BioAnalyzer 2100 Expert (Agilent Technologies, Santa Clara, CA).
Alginate-encapsulated follicles were placed in individual wells of a 96-well plate containing 100 ul of growth media (alpha minimum essential medium (alphaMEM) supplemented with 10 mIU/ml recombinant FSH (Organon, Roseland, NJ), 3 mg/ml bovine serum albumin (MP Biomedicals, Irvine, CA), 1 mg/ml bovine fetuin (Sigma, St. Louis, MO), 5 ug/ml insulin, 5 ug/ml transferrin and 5 ng/ml selenium) and cultured for 2, 4, 5, 6 or 8 days in a 5% CO2:21% O2 atmosphere. Half of the culture media (50 ul) was exchanged every 2 days and conditioned media stored at -80ºC. To collect follicles at each time point, alginate-encapsulated follicles were removed from growth media, pooled (20-40 follicles per group, n = 3 independent experiments) and transferred into 1 ml of Liebovitz L-15 media containing 10 U/ml alginate lyase (Sigma) for 20 min at 37ºC. Follicles were aspirated, then transferred into microcentrifuge tubes, flash frozen in liquid nitrogen and stored at -80ºC until subsequent RNA isolation was performed.