|
| Status |
Public on Jul 26, 2013 |
| Title |
Basal cells_Patient4 |
| Sample type |
RNA |
| |
|
| Source name |
Basal cells
|
| Organism |
Homo sapiens |
| Characteristics |
gender: Female
age: 26
cell type: Lin-CD49fhiEpCAMneg/low (basal stem/bipotent cell-enriched)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Human breast tissues obtained from reduction mammoplasty surgeries were collected, processed and single cells FACS purified as described by Eirew et al Nature Medicine (PMID 19029987).
RNA was isolated from approximately a million cells in Trizol (Invitrogen), re-purified using the RNeasy Micro kit (Qiagen), and total RNA quality was assessed with the Agilent 2100 bioanalyzer prior to microarray analysis. Samples with a RIN value of greater than or equal to 8.0 were deemed to be acceptable for microarray analysis.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from about 25ng RNA using Agilent One-Color Microarray-Based Exon Analysis Low Input Quick Amp WT Labeling v1.0 according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
600ng of Cy3-labelled cRNA (specific activity >15 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25ul containing 1ul Agilent fragmentation buffer and 5ul of 10X Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Human 8x60K Microarrays (Design ID 028004) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried for 1min in acetonitrile.
|
| Scan protocol |
Arrays were scanned with the Agilent DNA Microarray Scanner at a 3um scan resolution.
|
| Description |
Patient-4, Lin-CD49fhiEpCAMneg/low (basal stem/bipotent cell-enriched)
|
| Data processing |
Data was processed with Agilent Feature Extraction 10.10.
|
| |
|
| Submission date |
Apr 12, 2012 |
| Last update date |
Jul 26, 2013 |
| Contact name |
Connie Eaves |
| E-mail(s) |
ceaves@bccrc.ca
|
| Phone |
(+1) 604-675-8109
|
| Fax |
(+1) 604-877-0712
|
| URL |
http://www.terryfoxlab.ca/people/ceaves/connie.aspx
|
| Organization name |
BC Cancer Agency
|
| Department |
Terry Fox Laboratory
|
| Lab |
Eaves Lab
|
| Street address |
675 W 10th Ave
|
| City |
Vancouver |
| State/province |
BC |
| ZIP/Postal code |
V5Z 1L3 |
| Country |
Canada |
| |
|
| Platform ID |
GPL14550 |
| Series (1) |
| GSE37223 |
Global gene expresion profile of phenotypically distinct human mammary epithelial subpopulations of various developmental age |
|
