|
| Status |
Public on Jul 01, 2014 |
| Title |
Wild type technical replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
HCT116 wild type cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116
genotype/variation: Wild type
|
| Treatment protocol |
The wild-type HCT116 line was obtained from ATCC (ATCC number CCL-247). Isogenic BRCA2-deficient lines were generated by sequentially targeting both alleles of the BRCA2 gene by homologous recombination (J. Xian and C. Caldas).
|
| Growth protocol |
Cells were incubated in 6mm plates. HCT116 human colon carcinoma cells were maintained in McCoy’s 5A media (Gibco, Invitrogen) supplemented with 10 % fetal bovine serum (Sigma-Aldrich, St. Louis, MO) and 20 mM L-glutamine, at 37 C in a humidified atmosphere containing 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from frozen HCT116 samples (three wild type and three BRCA2-/-) using QIAzol™ lysis reagent (Qiagen, Maryland, USA). The tissues were homogenized in 800 µL QIAzol and transferred to Phase Lock Gel tubes (2 mL, heavy) (Eppendorf AG, Hamburg, Germany) to which 200 µL chloroform was added. Samples were centrifuged and the aqueous phase was removed and transferred to a new tube with an equal volume of isopropanol. After centrifugation, the supernatant was removed from the precipitated RNA pellets, and the pellets were washed with 70% ethanol. The air-dried RNA was dissolved in RNase/DNase free water and stored frozen at -80 °C. Total RNA samples were analyzed on a NanoDrop (Thermo Fisher Scientific, Waltham, MA) spectrophotometer to determine RNA quality and concentration. One µL of the RNA (was adjusted within a range of 25-500 ng/µL) was analyzed on the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) using the RNA 6000 Nano kit.
|
| Label |
biotin
|
| Label protocol |
Samples with a resulting total RNA of 1 µg or greater and with a RNA integrity number (RIN) of 7.0 or greater were used and the whole transcript sense target labeling procedure followed the Affymetrix protocol, including the RiboMinus™ rRNA removal step. Three technical replicates were compared for each of the genotypes (HCT116 homozygous BRCA2-/- and isogenic wild type) for a total of six experiments.
|
| |
|
| Hybridization protocol |
Labelled RNA was probed on Human Exon Array 1.0 ST chips (Affymetrix, Santa Clara, California) for gene expression analysis and scanned at the CTAG Facility of the British Columbia Cancer Agency, Vancouver, BC, Canada.
|
| Scan protocol |
Array scanning was performed according to the manufacturer's instructions (Affymetrix)
|
| Description |
HCT116 wild type cells
|
| Data processing |
The microarray data were analyzed using the oneChannelGUI package of the R statistical programming language (R version 2.11.1, R Development Core Team, 2010). Raw intensity calls were normalized using quantile normalization (Bolstad et al 2003) and probeset summarization (core plus extended) undertaken with RMA (Irizarry et al 2003)
probe group file: HuEx-1_0-st-v2.r2.pgf
meta-probeset file: HuEx-1_0-st-v2.r2.dt1.hg18.core.mps
|
| |
|
| Submission date |
Feb 10, 2014 |
| Last update date |
Jul 01, 2014 |
| Contact name |
Steven McKinney |
| E-mail(s) |
smckinney@bccrc.ca
|
| Phone |
604-675-8000
|
| Organization name |
British Columbia Cancer Agency
|
| Department |
Molecular Oncology
|
| Lab |
Samuel Aparicio
|
| Street address |
675 West 10th Ave
|
| City |
Vancouver |
| State/province |
BC |
| ZIP/Postal code |
V5Z 1L3 |
| Country |
Canada |
| |
|
| Platform ID |
GPL5175 |
| Series (1) |
| GSE54830 |
Up-regulation of IFN-related genes in BRCA2-/- cells |
|
