Strain: Bmp15-/- on B6/129 background; Gender:females; Age: 4-5 months old; Follicle stage: large antral follicles
Female WT, Bmp15-/-, and DM mice were primed with eCG (5IU/mouse, EMD Biosciences, Inc., Calbiochem, La Jolla, CA) for 48 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. After removing all of the denuded oocytes, cumulus cells were gathered together by gently swirling the dish, and were transferred into a 1.5 ml eppendorf centrifuge tube. The cells were finally spun down by gentle centrifugation, and the pellet was re-suspended in 350 µl of RLT buffer (Qiagen, Valencia, CA) after removing the supernatant. After brief vortexing, the cumulus cell lysate was snap frozen in liquid nitrogen and temporarily stored at -80°C until RNA isolation. Three sets of WT, Bmp15-/- and DM cumulus cell samples were collected and employed in microarray study. For each sample, about 75-100 COCs, obtained from 3-4 mice, were used for cumulus cell collection. Medium used for cumulus cells 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 cumulus cell isolation. Total RNA was extracted from cumulus cells using the RNeasy Micro Kit (Qiagen, Valencia, CA) according to the manufacturer’s instruction. The RNA quality and yield of each sample were determined using the Bioanalyzer 2100 and RNA 6000 Pico LabChip assay (Agilent Technologies Inc, Palo Alto, CA) in combination with Quant-iTTM RiboGreen Reagent according to supplied protocols (Invitrogen, Carlsbad, CA).
Ten nanograms of total RNA from each sample was employed in the two-round cDNA synthesis and subsequent in vitro-transcription according to the Two-Cycle Eukaryotic Target Labeling Assay (Affymetrix Expression Analysis Technical Manual: “ Section 2: Eukaryotic Sample and Array Processing” (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
Equal amounts, 15 µg, of fragmented and biotin-labeled cRNA from each sample were then hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 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).
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).
References cited Cui, X. and Churchill, G. A. (2003). Statistical tests for differential expression in cDNA microarray experiments. Genome Biol, Epub 2003 Mar 17. Cui, X., Hwang, J. T., Qiu, J., Blades, N. J. and Churchill, G. A. (2005). Improved statistical tests for differential gene expression by shrinking variance components estimates. Biostatistics 6, 59-75. Gautier, L., Cope, L., Bolstad, B. M. and Irizarry, R. A. (2004). affy--analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 20, 307-15. Irizarry, R. A., Bolstad, B. M., Collin, F., Cope, L. M., Hobbs, B. and Speed, T. P. (2003). Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res 31. Storey, J. D. and Tibshirani, R. (2003). Statistical significance for genomewide studies. Proc Natl Acad Sci U S A, Epub 2003 Jul 25. Wu H, K. M., Cui X, Churchill GA. (2003). MAANOVA: A Software Package for the Analysis of Spotted cDNA Microarray Experiments. In The Analysis of Gene Expression Data: An Overview of Methods and Software, (ed. G. E. Parmigiani G, Irizarry RA, Zeger SL), pp. 313-431. New York: Springer.
Probe level data were imported into the R software environment and expression values were summarized using the RMA (Robust MultiChip Average) function (Irizarry et al., 2003) in the R/affy package (Gautier et al., 2004). Using the R/maanova package (Wu H, 2003), an analysis of variance (ANOVA) model was applied to the data, and F1, F2, F3 and Fs test statistics were constructed along with their permutation p-values(Cui and Churchill, 2003; Cui et al., 2005). False discovery rate (FDR) (Storey and Tibshirani, 2003) was then assessed using the R/qvalue package to estimate q-values from calculated Fs test statistics. Three pair-wise comparison analyses: DM vs. WT, Bmp15-/- vs. WT, and DM vs. Bmp15-/-, were generated, and the significantly changed transcripts were identified using the criteria of FC (fold change) p < 0.01. Results were annotated using information provided by Affymetrix (12/20/2005 release).