|
| Status |
Public on Jul 08, 2015 |
| Title |
Primary tumor breast - BCPT0925 |
| Sample type |
RNA |
| |
|
| Source name |
Primary tumor breast
|
| Organism |
Homo sapiens |
| Characteristics |
sample group: validation
setnr: 3023,3057
case-control status: 0,0
tissue: Primary tumor breast
|
| Treatment protocol |
The fresh frozen tumor samples were collected from liquid nitrogen and cryosectioning was performed on a Cryo-Star HM 560 M. Five μm sections were cut from the surface of the same tumor sample that was used for isolation of RNA and DNA. The section was counterstained with hematoxylin and eosin and dehydrated through graded concentrations of ethanol to xylene before mounting. The percentage of tumor cells, as well as the percentage of epithelial cancer cells in relation to stroma, was quantified in the frozen tumor sections by a breast cancer pathologist.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Extraction of total RNA from frozen tumors was carried out using the Qiagen RNeasy Mini Kit (Qiagen, Germany). The integrity of the RNA extracts was tested on an Agilent 2100 Bioanalyzer (Agilent Technologies, Rockville, MD), measuring the 28S: 18S ribosomal RNA ratio. RNA extracts of high quality were stored at -70C until microarray analyses.
|
| Label |
biotin
|
| Label protocol |
Preparation of in vitro transcription (IVT) products and oligonucleotide array hybridization and scanning were performed according to the Affymetrix protocol (Santa Clara, CA, USA). In brief, the amount of starting total RNA for each probe preparation varied between 2 and 5 μg. IVT reactions were performed in batches to generate biotinylated cRNA targets, which were subsequently chemically fragmented at 95C for 35 min.
|
| |
|
| Hybridization protocol |
Fragmented and biotinylated cRNA (10 μg) was hybridized at 45C for 16 h to a high density oligonucleotide custom made Affymetrix array chip (Human Cancer G110 Array). The arrays were then washed, stained with streptavidin-phycoerythrin (SAPE, final concentration of 10 μg/ml).
|
| Scan protocol |
The arrays were scanned according to the manufacturer’s instructions (Affymetrix Genechip® Technical Manual, 2001). The scanned images were inspected for the presence of obvious defects (artifacts or scratches) on the array. In case of visible microarray artifacts, the sample was rehybridized and rescanned on new chips using the same fragmented probe.
|
| Description |
Some arrays correspond to multiple study subjects, which is indicated by the setnr/case-control status
|
| Data processing |
The microarray analysis was done in the open source software R using the aroma.affymetrix package. Each array was individually background corrected and normalized using robust multichip averaging (RMA). The arrays were then quantile normalized for comparability. Only perfect match (PM) probes were used in the normalization steps. The signals from multiple PM probes targeting the same transcript were summarized using a probe level model adjusting for chip effects. A variance filter was employed and probes with inter-quartile-range less than 0.5 were excluded, resulting in 28449 retained probes.
|
| |
|
| Submission date |
Jun 19, 2013 |
| Last update date |
Jul 08, 2015 |
| Contact name |
Alexandra Jauhiainen |
| E-mail(s) |
alexandra.jauhiainen@ki.se
|
| Organization name |
Karolinska Institutet
|
| Department |
Medical Epidemiology and Biostatistics
|
| Street address |
PO Box 281
|
| City |
Stockholm |
| ZIP/Postal code |
SE-171 77 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL10379 |
| Series (1) |
| GSE48091 |
A nested case-control study to investigate drivers for metastatic disease in breast cancer |
|
