|
| Status |
Public on Nov 19, 2015 |
| Title |
tumor_xenograft_10_SSEA4-_replicate1 |
| Sample type |
RNA |
| |
|
| Source name |
tumor_xenograft_10_SSEA4-
|
| Organism |
Homo sapiens |
| Characteristics |
host mouse strain: Hsd:Athymic Nude-Foxn1nu (athymic nude mice)
host mouse gender: female
xenograft tissue: human breast cancer
xenograft batch: 10
cell fraction: SSEA4-
|
| Treatment protocol |
Whole tumors were dissected out together with the surrounding stromal tissue and were placed in a 50 mL falcon tube containing MACS® tissue storage solution (Miltenyi Biotec). Tumor tissue was dissociatied using the Tumor Dissociation Kit, human (Miltenyi Biotec). SSEA4+ and SSEA4- tumor cell subpopulations were isolated by magnetic activated cell sorting (MACS®). After dissociation and depletion of mouse cells, the cells were resuspended in PEB buffer (PBS, pH 7.2, 0.5% bovine serum albumin, and 2 mM EDTA; prepared by diluting MACS BSA Stock Solution 1:20 with autoMACS® Rinsing Solution) at a concentration of 1x10E7 cells per 100 µl. After adding 10 µl SSEA4-PE (Miltenyi Biotec) per 100 µl the suspension was incubated at 4°C under continuous agitation (MACSmix Tube Rotator, Miltenyi Biotec) for 10 min. Then the cells were pelleted at 300 g for 10 min, resuspended in 80 µl PEB per 1x10E7 cells, 20 µl of anti-PE MicroBeads (Miltenyi Biotec) were added and incubated at 4°C under continuous agitation for 15 min. Cells were washed in 1 ml PEB per 1x10E7 cells, pelleted and resuspended in 500 µl PEB per separation. SSEA4+ tumor cells were isolated using an MS-column (Miltenyi Biotec), SSEA4- tumor cells were isolated using an LD-column (Miltenyi Biotec). Purity of the isolated cells was evaluated using flow cytometry.
|
| Growth protocol |
Human breast cancer xenografts were established from patient’s primary tumor surgical specimens, by grafting tumor fragments into the interscapular fat pad of athymic nude mice, and maintained through in vivo passages as previously described (PMID: 17606733).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the miRNeasy Kit (QIAGEN).
|
| Label |
Cy3
|
| Label protocol |
100 ng of each RNA was used as template to produce Cy3- labeled cRNA. The RNA samples were amplified and labeled using the Low Input Quick Amp Labeling Kit (Agilent Technologies) following the manufacturer’s protocol.
|
| |
|
| Hybridization protocol |
Cy3 labeled cRNAs were hybridized overnight (17 hours, 65°C) to Agilent Whole Human Genome Oligo Microarrays 8x60K v2 (Design ID 039494) using Agilent’s recommended hybridization chamber and oven.Finally, microarrays were washed once with Agilent Gene Expression Wash Buffer 1 for 1 min at room temperature followed by a second wash with preheated Agilent Gene Expression Wash Buffer 2 (37 °C) for 1 min.
|
| Scan protocol |
Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent’s Microarray Scanner System (G2505C, Agilent Technologies, Palo Alto, USA).
|
| Description |
10_neg_1
|
| Data processing |
The Agilent Feature Extraction Software (FES 10.7.3.1 ) was used to read out and process the microarray image files.
|
| |
|
| Submission date |
May 15, 2014 |
| Last update date |
Nov 19, 2015 |
| Contact name |
Stefan Tomiuk |
| E-mail(s) |
stefant@miltenyibiotec.de
|
| Organization name |
Miltenyi Biotec GmbH
|
| Department |
Bioinformatics
|
| Street address |
Friedrich-Ebert-Str. 68
|
| City |
Bergisch-Gladbach |
| ZIP/Postal code |
51429 |
| Country |
Germany |
| |
|
| Platform ID |
GPL17077 |
| Series (2) |
| GSE57703 |
SSEA4 is a marker for chemotherapy resistance [mRNA] |
| GSE57705 |
SSEA4 is a marker for chemotherapy resistance |
|
