21-day old (For Experiment 1) female Marf1+/+ (WT) and Marf1 Mutant mice were primed with eCG (5IU/mouse, EMD Biosciences, Inc., Calbiochem, La Jolla, CA) for 46 hours to stimulate follicle development. Cumulus-oocyte complexes (COCs) were released by puncturing large antral follicles on ovarian surface with a pair of 26 gauge needles. Released COCs were collected and washed for three times by passing through 3 dishes of medium. Cumulus cells were then stripped from the oocyte by passing COCs several times through a hand-drawn small fine glass pipette with an inner diameter slightly narrower than the oocyte. The resulting denuded fully-grown oocytes (FGOs) were then collected into a 1.5 ml eppendorf centrifuge tube and re-suspended in 350 µl of RLT buffer (Qiagen, Valencia, CA). After brief vortexing, the oocyte lysate was snap frozen in liquid nitrogen and temporarily stored at -80°C until RNA isolation. Growing oocytes (Experiment2) were isolated from 12-d old mice by digesting the whole ovaries with 4 mg/ml of collagenase, type I (Worthington Biochemical Corporation, Lakewood, NJ) and 0.02 mg/ml of deoxyribonuclease I in culture medium. The dissociated oocyte-granulosa cells complexes were then transferred into Ca2+, Mg2+-free PBS containing 1 mg/ml of collagenase and digested for about 20 min to remove the granulosa cells. Growing oocytes (GOs) were then collected into a 1.5 ml eppendorf centrifuge tube and re-suspended in 350 µl of RLT buffer (Qiagen, Valencia, CA). After brief vortexing, the oocyte lysate was snap frozen in liquid nitrogen and temporarily stored at -80°C until RNA isolation. Three sets of Marf1+/+ (WT) and Marf1 Mutant FGO and GO samples were collected and employed in microarray study. Medium used for oocyte isolation was MEM-α (Invitrogen Corporation, Grand Island, NY) supplemented with 3 mg/ml of crystallized lyophilized bovine serum albumin (Sigma, St. Louis, MO), 75 mg/liter of penicillin G (Sigma) and 50 mg/liter streptomycin sulfate (Sigma). Milrinone (Sigma), a selective inhibitor of oocyte-specific phosphodiesterase (PDE3), was added into the medium at the concentration of 5 µM to prevent the GV-stage oocytes from undergoing maturation during the process of COC and FGO.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from oocytes using the PicoPure RNA isolation kit according to the manufacturer's instruction. The quality and quantity of RNA were measured by the Bioanalyzer 2100 and RNA 6000 Pico LabChip assay (Agilent Technologies Inc, Palo Alto, CA) in combination with Quant-iT™ RiboGreen Reagent according to supplied protocols (Invitrogen, Carlsbad, CA).
Label
biotin
Label protocol
RNA samples were subjected to cDNA synthesis and amplification using the Nugen Ovation Pico WTA System (NuGEN Technologies, Inc, San Carlos, CA). The resulting cDNAs were then fragmented and biotin-labeled using the Encore™ Biotin Module (NuGEN Technologies, Inc).
Hybridization protocol
Fragmented and biotin-labeled cDNA from each sample were then hybridized to Affymetrix GeneChip® Mouse Gene 1.0 ST arrays for 16 h at 45°C. Post-hybridization staining and washing were performed according to manufacturer's protocols using the Fluidics Station 450 instrument (Affymetrix
Scan protocol
The arrays were scanned with a GeneChip™3000 laser confocal slide scanner (Affymetrix) and the images were quantified using Gene Chip Operating Software version 1.2 (GCOS, Affymetrix).
Description
Transcriptional silent oocytes
Data processing
After the post-hybridization staining and washing, the arrays were scanned with a GeneChip™3000 laser confocal slide scanner (Affymetrix) and the images were quantified using Gene Chip Operating Software version 1.2 (GCOS, Affymetrix). Average signal intensities for each probe set within arrays were calculated by and exported from Affymetrix’s Expression Console (Version 1.1) software using the RMA method which incorporates convolution background correction, summarization based on a multi-array model fit robustly using the median polish algorithm, and sketch-quantile normalization. For this experiment, one pairwise comparison was used to statistically resolve gene expression differences between Type levels using the R/maanova analysis package40 (Wu et al. 2003). Specifically, differentially expressed genes will be detected by using Fs, a modified F-statistic incorporating shrinkage estimate of variance components from within the R/maanova package40-41. Statistical significance levels of the pairwise comparison wase calculated by permutation analysis (1000 permutations) and adjusted for multiple testing using the false discovery rate (FDR), q-value, method42. Differentially expressed genes are declared at an FDR q-value threshold of 0.05.
