strain: CD-1 tissue: Multi-layered secondary follicles (150-180 µm in diameter) mechanically isolated from ovaries of 16-day-old mice age: 16-day-old mice
At each time point, follicles were pooled (for the microarray, 20-40 follicles per group, n = 3 independent experiments) and transferred into 1 ml of Liebovitz L-15 medium containing 10 U/ml alginate lyase (Sigma) for 20 min at 37°C to remove them from alginate. Follicles were aspirated, transferred into microcentrifuge tubes, flash frozen in liquid nitrogen, and stored at -80°C until RNA isolation was performed.
Multi-layered secondary follicles (150-180 µm in diameter) were mechanically isolated from ovaries of 16-day-old mice and individually encapsulated in alginate (FMC BioPolymers, Philadelphia, PA) as previously described (Kreeger et al. , 2006, West-Farrell et al., 2009, Xu et al. , 2006a). Alginate-encapsulated follicles were placed in individual wells of a 96-well plate containing 100 µl of growth medium (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 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml selenium and cultured for 2, 4, 5, 6, or 8 days for microarray analysis. Half of the culture medium was exchanged every 2 days.
RNA was purified from follicles using the Qiagen RNeasy Micro Kit according to the manufacturer’s protocol (Qiagen, Valencia, CA).
mRNA samples were in vitro labeled using the TargetAmp 1-Round Aminoallyl-aRNA Kit (Epicentre, Madison, WI) a
randomly hybridized to BeadChips
Raw signal intensities of each probe were obtained using BeadStudio (Illumina).
TargetAmp 1-Round Aminoallyl-aRNA Kit 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). REF0-3
Statistical analyses were done in R (2008). Inadequate Illumina microarray probes due to misannotations or intronic coverage were removed from analysis using the Mouse WG V2.0 R0 file within ReMOAT (http://remoat.sysbiol.cam.ac.uk) (Barbosa-Morais et al. , 2010). Adequately annotated probes were considered to be above background if at least 2 of the 3 replicates measured were above background at P ≤ 0.01. The data were transformed using the variance stabilization transformation method (Lin et al. , 2008) and normalized by robust spline normalization (Du et al. , 2008).