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Sample GSM173616 Query DataSets for GSM173616
Status Public on Oct 22, 2007
Title wildtype cumulus cells #1
Sample type RNA
 
Source name Cumulus cells from PMSG (48h) primed adult wildtype ovarian follicles
Organism Mus musculus
Characteristics Strain: wildtype on B6/129 background; Gender: females; Age: 4-5 months old; Follicle stage: large antral follicles
Extracted molecule total RNA
Extraction protocol 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).
Label Biotin
Label protocol 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).
 
Hybridization protocol 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).
Scan protocol 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).
Description 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.
Data processing 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).
 
Submission date Mar 07, 2007
Last update date Aug 28, 2018
Contact name You-Qiang Su
E-mail(s) youqiang.su@jax.org
Organization name The Jackson Laboratory
Street address 600 Main Street
City Bar Harbor
State/province ME
ZIP/Postal code 04609
Country USA
 
Platform ID GPL1261
Series (1)
GSE7225 Identification and characterization of the changed transcripts in cumulus cells of Bmp15-/- and Bmp15-/-Gdf9+/--DM mice.
Relations
Reanalyzed by GSE119085

Data table header descriptions
ID_REF
VALUE RMA

Data table
ID_REF VALUE
1415670_at 9.427288
1415671_at 8.635649
1415672_at 10.641341
1415673_at 7.474331
1415674_a_at 8.519612
1415675_at 10.026134
1415676_a_at 10.920104
1415677_at 8.048604
1415678_at 12.012173
1415679_at 10.093927
1415680_at 8.458998
1415681_at 8.307484
1415682_at 9.183236
1415683_at 9.949656
1415684_at 9.245783
1415685_at 9.318491
1415686_at 11.563532
1415687_a_at 10.23868
1415688_at 10.674031
1415689_s_at 6.745625

Total number of rows: 45101

Table truncated, full table size 898 Kbytes.




Supplementary file Size Download File type/resource
GSM173616.CEL.gz 3.8 Mb (ftp)(http) CEL
Raw data provided as supplementary file
Processed data included within Sample table

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