|
| Status |
Public on Jun 15, 2012 |
| Title |
NM-3 |
| Sample type |
RNA |
| |
|
| Source name |
NOV_116G
|
| Organism |
Homo sapiens |
| Characteristics |
age: 51
cancer history: No
carrier: No
familial mutation: Not Applicable
Stage: Not Applicable
grade: Not Applicable
cell origin: Morphologically normal ovarian surface epithelium cells
culture medium: OSE
passage: 13
class: NM
|
| Biomaterial provider |
Fonds de la recherche en santé du Québec (FRSQ) Cancer Research Network (RR-Cancer) Tumor Bank of the Centre hospitalier de l’Université de Montréal (CHUM)
|
| Treatment protocol |
Morphologically normal surface epithelium cells were obtained by gently scraping, 2-3 times with a rubber scraper, the ovarian surface epithelium of the surgical specimen provided by the pathologist. The cells collected were then seeded in standard OSE medium consisting of 50:50 medium 199:105 (Sigma) supplemented with 15% fetal bovine serum (FBS), 2.5 µg/mL amphotericin B and 50 µg/mL gentamicin.
|
| Growth protocol |
After rinsing, an explant of the surgical specimen was placed in 35-mm culture dishes, and held surface down so that the ovarian surface epithelium was maintained in a small amount of medium in contact with the bottom of the culture dishes, allowing the NOSEs to colonize the plastic. The cells were then passaged at 80% of confluence. A subgroup of NOSEs was cultivated in a modified medium (MM) i.e. the standard OSE medium supplemented with epidermal growth factor, hydrocortisone, insulin and bovine pituitary extract. Finally, tumors of ovaries (TOVs) were established in OSE medium supplemented with 10% FBS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with TRIzolTM reagent (Gibco/BRL, Life Technologies Inc., Burlington, ON, Canada). RNA was extracted directly from homogenized cells grown to 80% confluence in 100-mm Petri dishes. RNA quality was monitored with a 2100 Bioanalyzer and RNA 6000 Nano LabChip kit (Agilent Technologies, Missisauga, Ontario, Canada) according to the manufacturer’s protocol.
|
| Label |
Biotin
|
| Label protocol |
Reverse Transcription was performed on 1 µg of total RNA with the SuperScriptTM First-Strand Synthesis System (Invitrogen Canada Inc., Burlington, Ontario, Canada) and random hexamers, according to the procedure recommended by the manufacturer. c-DNA samples were diluted at 1/3 or 1/6 in water prior to q-RT-PCR. Three µl of this dilution were used for each experiment. An in vitro transcription was performed on this cDNA and the resulting cRNA was biotinylated via incorporation of biotinylated dUTP and dCTP. DNA from samples was fragmented in 40mM Tris acetate, 100mM potassium acetate, and 30mM MgCl2 (pH 8.1) at 951C in order to reduce secondary structure.
|
| |
|
| Hybridization protocol |
A measure of 15 mg of cRNA was hybridized to an Affymetrix HuGeneFL microarray. Following hybridization, the arrays were washed and stained with streptavidin-phycoerythrin.
|
| Scan protocol |
Microarrays were scanned with a Hewlett Packard Gene Array scanner.
|
| Data processing |
Gene expression data were corrected for background using the Robust Multiarray Averaging (RMA) package, normalized using the Quantile method and summarized using the Median Polish method. Differentially-expressed probesets (DEPS) were identified on the basis of a moderated t-statistic computed with the Bioconductor Limma package for each contrast of two a priori classes of samples. The Benjamini and Hochberg (BH) correction method was applied to control the false discovery rate across all genes. DEPS retained as candidates correspond to probesets with q-value (adjusted BH p-values) ≤ 0.05 for most of the comparisons with the exception of the M1/2 vs. TM1/2 contrast where q-value was ≤ 0.2.
|
| |
|
| Submission date |
Jan 08, 2012 |
| Last update date |
Jun 15, 2012 |
| Contact name |
Diala Abed-Rabbo |
| E-mail(s) |
diala.abd.rabbo@umontreal.ca
|
| Organization name |
Université de Montréal
|
| Street address |
C.P. 6128, succursale Centre-ville
|
| City |
Montréal |
| ZIP/Postal code |
H3C 3J7 |
| Country |
Canada |
| |
|
| Platform ID |
GPL80 |
| Series (1) |
| GSE34928 |
Gene Expression in Normal and Tumor Ovarian Epithelial Cells from Non-carriers and Heterozygous Carriers of a BRCA French Canadian Mutation. |
|
