|
| Status |
Public on Jul 25, 2014 |
| Title |
MCF7-hypoxia-2 |
| Sample type |
RNA |
| |
|
| Source name |
MCF7-hypoxia-0.1%O2 48h
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF7
treatment: hypoxia
|
| Treatment protocol |
A hypoxic incubator (Coy Laboratory Hypoxic workstation glove box) was used to expose cells to continuous controlled hypoxic conditions. This humidified, temperature controlled (37 ºC) chamber supplemented with 5% CO2, and N2 (as required to maintain controlled O2 levels). Cells were exposed to 0.1% O2 levels for 48 h. Normoxic controls were incubated in parallel in a humidified incubator supplemented with 5% CO2 at 37 ºC. MCF7 cells were seeded at 50 000 cells per well in 24-well plates (1.9 cm2) and grown for 24 h. Cells were transfected with 20 nM siRNA duplexes (Shanghai GenePharma Co., Ltd, China), using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer’s protocol. A second transfection was carried out after 24 h following the same protocol. Cells were harvested 24 h after the second transfection and used for RNA and protein extraction.
|
| Growth protocol |
Breast cancer cell line MCF7 was maintained in RPMI 1640 (Invitrogen) medium supplemented with 10% foetal bovine serum (FBS) (Bovigen), in polystyrene flasks. Cells were maintained at 37 ºC with 5% CO2. All experiments were conducted in triplicate with independent cell cultures.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRIzol reagent (Invitrogen) following the manufacturers protocol. RNA quantity and quality were determined using a Nanodrop-8000 spectrophotometer (Nanodrop Technology) and Agilent 2100 Bioanalyzer.
|
| Label |
biotin
|
| Label protocol |
100-300 ng of total RNA was used to synthesise double stranded cDNA using random hexamers. The cDNA was then amplified to produce antisense cRNA, which was then reverse transcribed in a second cycle of cDNA synthesis. The second cycle incorporates dUTP into the cDNA sequence, which allows it to be fragmented using uracil DNA glycosylase and apurinic/apyrimidic endonuclease I.
|
| |
|
| Hybridization protocol |
Following biotinylation, these fragments were hybridised overnight to a Affymetrix miRNA 3.1 array
|
| Scan protocol |
The arrays were then washed, stained using a fluorescently-labelled antibody, and scanned using a high-resolution scanner (GeneChip® Scanner 3000 7G). Affymetrix® GeneChi p® Command Console® (AGCC) Software
|
| Description |
mammary gland/breast; derived from metastatic site: pleural effusion
Mature miRNA and precursor miRNA expression data
|
| Data processing |
Intensity data were analysed using Partek® software (Partek Inc.). Data were normalised by quantile normalisation and log 2 transformed. Differential expression was determined by ANOVA and corrected for false discovery.
|
| |
|
| Submission date |
Aug 19, 2013 |
| Last update date |
Jul 25, 2014 |
| Contact name |
Kanchana Veronika Bandara |
| Organization name |
Flinders University
|
| Department |
Renal Department
|
| Street address |
Flinders Drive
|
| City |
Bedford Park |
| State/province |
SA |
| ZIP/Postal code |
5042 |
| Country |
Australia |
| |
|
| Platform ID |
GPL16384 |
| Series (1) |
| GSE49999 |
Precursor and mature microRNA expression in MCF7 cells in hypoxia |
|
