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Status |
Public on Aug 29, 2011 |
Title |
v-ras-Ha transformed keratinocytes without IL1ra, rep3 |
Sample type |
RNA |
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Source name |
v-ras-Ha transformed keratinocytes with PBS treatment
|
Organism |
Mus musculus |
Characteristics |
genotype: wild type developmental stage: newborn tissue: epidermis cell type: v-ras-Ha-transduced keratinocytes treatment: PBS
|
Treatment protocol |
Primary mouse keratinocytes and hair follicle buds were isolated from newborn epidermis from wild type mice. Keratinocytes were adenovirus infected for 30 min with serum-free medium with a multiplicity of infection of 10 viral particles/cell and 4 ug/ml of Polybrene (Sigma) to enhance uptake. Five hours post v-rasHa-transduction, interleukin-1-receptor antagonist, Anakinra (Amgen, Inc, Thousand Oaks, CA), was added to the keratinocytes cultures at a final concentration of 5 ug/ml . Cultures were kept for an extra 3 days but IL-1Ra was replaced after 2 days. Thereafter, RNA from IL1R antagonist- or PBS-treated cultures was prepared.
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was harvested from cultured v-rasHa-transduced keratinocytes in the presence of PBS or Anakinra. Total RNA was extracted using Trizol reagent according to the manufacturer’s instructions. RNA was purified using the RNAeasy Min-elute (Qiagen) RNA isolation kit. Total RNA samples were quantitated by UV spectrophotometry (OD260/280). Quality of the total RNA was assessed by using an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA).
|
Label |
Alexa-555
|
Label protocol |
First and second strand cDNA was prepared from the total RNA samples. cRNA target was prepared from the DNA template and verified on the Bioanalyzer.
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|
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Hybridization protocol |
cRNA was fragmented to uniform size and hybridized to Agilent Whole Genome 4x44K arrays.
|
Scan protocol |
Slides were washed and scanned on an Agilent G2565 Microarray Scanner.
|
Description |
Control_9
|
Data processing |
The gProcessSignal of Agilent Feature Extraction data was normalized using a global median-based method and filtered by the Agilent feature extraction flag “gIswellAboveBG” to eliminate bad probes with signals consistently lower than background across the samples. All of these procedures were done with customized R scripts (www.r-project.org).
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Submission date |
Aug 05, 2010 |
Last update date |
Aug 29, 2011 |
Contact name |
Rosalba Salcedo |
E-mail(s) |
lmirosi@mail.ncifcrf.gov
|
Phone |
301-846-6274
|
Fax |
301-846-6641
|
Organization name |
SAIC-NCI
|
Street address |
1050 Boyles Street, Bldg. 567
|
City |
Frederick |
State/province |
MD |
ZIP/Postal code |
21701 |
Country |
USA |
|
|
Platform ID |
GPL4134 |
Series (1) |
GSE22777 |
Autocrine IL-1α mediates NF-κB activation by oncogenic ras in keratinocytes |
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