|
| Status |
Public on Jul 29, 2009 |
| Title |
late passage keratinocytes from Casp-8F/–K5-Cre mouse and Casp-8F/+K5-Cre mouse (pair2). |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Epidermal keratinocytes isolated from the skin of Casp-8F/–K5-Cre mouse (3rd passage).
|
| Organism |
Mus musculus |
| Characteristics |
age: P3
tissue: epidermal keratinocytes
passage: 3
genotype: Casp-8F/–K5-Cre
|
| Treatment protocol |
Skin was removed from the mice and incubated overnight at 4 °C in 2.4 U/ml Dispase (Roche) to separate the epidermis from the dermis. The keratinocytes were then dissociated by incubation of the epidermis for 10 min in 0.1% trypsin, recovered by centrifugation, and plated on tissue-culture dishes in keratinocyte serum-free medium containing 50 µg/ml bovine pituitary extract and 5 ng/ml epidermal growth factor (Gibco-Invitrogen) supplemented with antibiotics. For passaging, cells were detached by incubation in PBS containing 0.1% trypsin, 0.5 mM EDTA and 0.1% glucose, and replated in keratinocyte serum-free medium.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with the RNeasy mini kit from QIAGEN as directed by the manufacturer.
|
| Label |
Cy3
|
| Label protocol |
cRNA-synthesis was performed with the “Low RNA Input Linear Amplification Kit PLUS, Two-Color” (#5188-5340, Agilent Technologies) as directed by the manufacturer.
|
| |
|
| Channel 2 |
| Source name |
Epidermal keratinocytes isolated from the skin of Casp-8F/+K5-Cre mouse (1st passage).
|
| Organism |
Mus musculus |
| Characteristics |
age: P3
tissue: epidermal keratinocytes
passage: 3
genotype: Casp-8F/+K5-Cre
|
| Treatment protocol |
Skin was removed from the mice and incubated overnight at 4 °C in 2.4 U/ml Dispase (Roche) to separate the epidermis from the dermis. The keratinocytes were then dissociated by incubation of the epidermis for 10 min in 0.1% trypsin, recovered by centrifugation, and plated on tissue-culture dishes in keratinocyte serum-free medium containing 50 µg/ml bovine pituitary extract and 5 ng/ml epidermal growth factor (Gibco-Invitrogen) supplemented with antibiotics. For passaging, cells were detached by incubation in PBS containing 0.1% trypsin, 0.5 mM EDTA and 0.1% glucose, and replated in keratinocyte serum-free medium.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with the RNeasy mini kit from QIAGEN as directed by the manufacturer.
|
| Label |
Cy5
|
| Label protocol |
cRNA-synthesis was performed with the “Low RNA Input Linear Amplification Kit PLUS, Two-Color” (#5188-5340, Agilent Technologies) as directed by the manufacturer.
|
| |
|
| |
| Hybridization protocol |
cRNA fragmentation, hybridization and washing steps were performed exactly as recommended by the manufacturer “Two-Color Microarray-Based Gene Expression Analysis Protocol V5.0.1” (see http://www.agilent.com for details).
|
| Scan protocol |
Slides were scanned on the Agilent Micro Array Scanner G2505 B at two different PMT settings, namely 100% and 10%, to increase the dynamic range of the measurements.
|
| Description |
mRNA expression differences between late passage keratinocytes from Casp-8F/–K5-Cre mouse and Casp-8F/+K5-Cre mouse (pair2).
|
| Data processing |
Data extraction and normalization were performed with the “Feature Extraction Software V9.1.3.1” by using the recommended default extraction protocol file: GE2-v5_91_0806.xml. Data from qualitatively suboptimal spots were flagged and excluded from further analysis if (i) CVsignal mean > 0.25, or (ii) SMglobal background corrected / SMlocal background corrected > 1.5, where 'CVsignal mean' is the coefficient of variance deduced from signal values for all pixels of a spot, and 'SMglobal background corrected / SMlocal background corrected' is the ratio of a global background-corrected signal mean value of a spot divided by its local background-corrected signal mean value.
|
| |
|
| Submission date |
Jul 27, 2009 |
| Last update date |
Jul 28, 2009 |
| Contact name |
Oliver Dittrich-Breiholz |
| E-mail(s) |
dittrich.oliver@mh-hannover.de
|
| Organization name |
Medical School Hannover
|
| Department |
Research Core Unit Genomics
|
| Street address |
Carl-Neuberg-Str. 1
|
| City |
Hannover |
| ZIP/Postal code |
30625 |
| Country |
Germany |
| |
|
| Platform ID |
GPL4134 |
| Series (2) |
| GSE17345 |
Analysis of temporal mRNA expression changes in cultured keratinocytes from Casp-8F/–K5-Cre and Casp-8F/+K5-Cre mice |
| GSE17365 |
Analysis of mRNA expression differences in the skin of Casp-8F/–K5-Cre and Casp-8F/+K5-Cre mice |
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