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Status |
Public on May 09, 2011 |
Title |
3325K6A_TVA |
Sample type |
RNA |
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|
Source name |
mouse mammary tumor, K6-tva RCAS-PyMT model
|
Organism |
Mus musculus |
Characteristics |
background: FVB/N tissue: mammary tumor transgenic: K6-tva RCAS-PyMT
|
Growth protocol |
The K6-tva BAC transgenic line was generating using the recombineering method. Briefly, the RP23-473O22 BAC clone (200-kb) containing the K6a gene was purchased from BACPAC Resources Center (Children’s Hospital Oakland Research Institute, Oakland, California). The Counter Selection BAC Modification Kit (Gene Bridges GmbH, Dresden, Germany) was used to modify the BAC following the manufacturer’s instructions. All mice were on the FVB/N background, and were kept on 2920X Teklad Global Extruded Rodent Diet (Soy Protein-Free) (Harlan Laboratories, Indianapolis, Indiana). All procedures using mice were performed in compliance with an Institutional Animal Care and Use Committee-approved animal protocol.
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Extracted molecule |
total RNA |
Extraction protocol |
All samples were collected fresh and snap-frozen in liquid nitrogen. Frozen samples were ground into fine powder in liquid nitrogen and RNA was extracted by Trizol (Lift Technologies) with an additional step of the isopropanol precipitation following the regular isopropanol precipitation step recommended in the protocol. This extra step cleans up the potential chemical contamination in the Trizol reagents.
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Label |
biotin
|
Label protocol |
Double-strand cDNA was synthesized from 5 µg of total RNA for each sample, and an in vitro transcription reaction was carried out to produce biotin-labeled cRNA from the cDNA. One-cycle target labeling and control reagents kit from Affymetrix (Part #900493) (Santa Clara, CA, USA) was used for the cDNA synthesis and biotin-labeling.
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Hybridization protocol |
Total of 15 µg biotin-labeled cRNA was fragmented, and 10 µg of the fragmented cRNA was prepared in the hybridization cocktail in a total volume of 200 µl, and 6.5 µg of them in 130 µl volume was hybridized onto the M430A 2.0 GeneChip in an Affymetrix Hybridization Oven 640 for 16-19 hours. We used Affymetrix recommended procedures for prehybridization, hybridization, washing, and staining with streptavidin-phycoerythrin (SAPE). Antibody amplification was employed with a biotin-linked antibody to streptavidin (Vector Laboratories, Burlingame, CA).
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Scan protocol |
After automated staining and washing (Affymetrix protocol Midi_euk2v3 450) using the GeneChip Fluidics Station 450, the arrays were scanned by the Affymetrix GeneChip scanner 3000 using the GeneChip operating system (GCOS) v1.2.
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Description |
Mouse mammary tumor, K6-tva RCAS-PyMT model. YL6-H33__3325K6A_TVA_PyMT_MG_430A_2.0
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Data processing |
A low level quantification was done in the GCOS1.2, and the probe level data (CEL file) was saved for each array and analyzed subsequently by DNA chip analyzer (dChip v2006).
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Submission date |
Mar 03, 2010 |
Last update date |
May 09, 2011 |
Contact name |
Chad Creighton |
E-mail(s) |
[email protected]
|
Organization name |
Baylor College of Medicine
|
Department |
Biostatistics, Ducan Cancer Center
|
Street address |
One Baylor Plaza, Mail Stop: BCM305
|
City |
Houston |
State/province |
TX |
ZIP/Postal code |
77030 |
Country |
USA |
|
|
Platform ID |
GPL8321 |
Series (1) |
GSE20614 |
Gene expression profiling of mouse tumors arising from Keratin 6-positive mammary cells |
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