|
| Status |
Public on Jul 29, 2011 |
| Title |
miRNA CEPI 489 |
| Sample type |
RNA |
| |
|
| Source name |
Laser captured microdissected ovarian cancer epithelial cells
|
| Organism |
Homo sapiens |
| Characteristics |
tumor stage/grade: IV/3
age at surgery: 48
|
| Treatment protocol |
Following pathological verification, a serous papillary epithelial cancer sample was embedded in cryomatrix (Shandon). Seven micron frozen sections were cut and attached to uncharged microscope slides for each sample. Immediately following dehydration and staining (HistoGene, LCM Frozen Section Staining Kit, Arcturus), slides were processed in an Autopix‘ instrument (Arcturus) for laser capture microdissection (LCM) of cells to CapSure Macro LCM Caps. Approximately 30,000 epithelia were collected from each cancer sample.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Fresh frozen tissues from tumors were cut into seven-micron sections, applied to non-charged slides, then fixed in 75% ethanol for 30 seconds, stained and dehydrated using the HistoGene LCM Frozen Section Staining Kit (Arcturus, Mountain View, CA). LCM was performed with an AutoPix Automated Laser Capture Microdissection System using the CapSure Macro Caps (Arcturus, Mountain View, CA). Approximately 10,000 cells were captured on each of 5-6 caps per sample. MiRNA was extracted from captured cells using the mirVana miRNA Isolation Kit (Ambion, Austin, TX).
|
| Label |
biotin
|
| Label protocol |
The 3’ ends of the RNA molecules were tailed and biotin-labeled using the mirVana™ miRNA Labeling Kit (Ambion Inc., Austin, TX). The kit’s dNTP mixture in the tailing reaction was replaced with a proprietary nucleotide mixture containing biotin-modified nucleotides (PerkinElmer, Waltham, MA).
|
| |
|
| Hybridization protocol |
Hybridization was performed according to Affymetrix-recommended procedures, except that the 20X GeneChip Eukaryotic Hybridization Control cocktail was omitted from the hybridization.
|
| Scan protocol |
Scanning was performed according to Affymetrix-recommended procedures. The fluorecence data is extracted at 1.4 micron pixel resolution using the Affymetrix 3000 7G scanner
|
| Description |
Serous ovarian tumor tissues were placed in cryotubes and immediately (<1 minute) frozen in liquid nitrogen following resection.
|
| Data processing |
For each probe, an estimated background value is subtracted that is derived from the median signal of a set of G-C-matched anti-genomic controls. Arrays within a specific analysis experiment were normalized together according to the variance stabilization method described by Huber et al. (Huber et al., 2002). Detection calls were based on a Wilcoxon rank-sum test of the miRNA probe signal compared to the distribution of signals from GC-content matched anti-genomic probes. A two-sample t-Test, with assumption of equal variance, was applied for statistical hypothesis testing. This test defined which probes are considered to be significantly differentially expressed based on a p-value of 0.01 and log2 difference ≥ 1.
|
| |
|
| Submission date |
Aug 02, 2010 |
| Last update date |
Nov 18, 2013 |
| Contact name |
Shubin Shahab |
| E-mail(s) |
shubin.shahab@gmail.com
|
| Organization name |
Georgia Institute of Technology
|
| Department |
Biology
|
| Lab |
McDonald
|
| Street address |
310 Ferst Drive
|
| City |
Atlanta |
| State/province |
GA |
| ZIP/Postal code |
30332 |
| Country |
USA |
| |
|
| Platform ID |
GPL9735 |
| Series (2) |
| GSE23383 |
miRNAs in ovarian cancer: A systems approach (miRNA data) |
| GSE23392 |
miRNAs in ovarian cancer: A systems approach |
|
| Relations |
| Reanalyzed by |
GSE52459 |
