|
| Status |
Public on Aug 23, 2014 |
| Title |
Trif_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
IEC isolated from healthy 28-day-old Trif LPS/LPS mice, biological replicate 2
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BBL6
genotype: Trif mut
age: 28-day-old
|
| Treatment protocol |
none
|
| Growth protocol |
IECs were prepared as described recently (Lotz et al., J. Exp Med. 2006). In brief, mouse intestinal tissue was incubated for 10 min at 37°C in 0.3 M EDTA. shaken vigorously and epithelial cells were separated from the underlying tissue and immune cells by repeated sedimentation at 1 x g at 4°C. Flow cytometric analysis revealed less than 5% of CD45+ cells in IEC preparations.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using TRIzol (Invitrogen, Darmstadt, Germany) according to the manufacturer’s protocol.
|
| Label |
Cy3
|
| Label protocol |
Synthesis of Cy3-labeled cRNA was performed with the “Quick Amp Labeling kit, one color” (#5190-0442, Agilent Technologies) according to the manufacturer’s recommendations.
|
| |
|
| Hybridization protocol |
cRNA fragmentation, hybridization and washing steps were also carried-out exactly as recommended: “One-Color Microarray-Based Gene Expression Analysis Protocol V5.7”.
|
| Scan protocol |
Slides were scanned on the Agilent Micro Array Scanner G2565CA (pixel resolution 5 µm, bit depth 20).
|
| Data processing |
Data extraction was performed with the “Feature Extraction Software V10.7.3.1” by using the recommended default extraction protocol file: GE1_107_Sep09.xml. Processed intensity values of the green channel (“gProcessedSignal” or “gPS”) were normalized by global linear scaling: All gPS values of one sample were multiplied by an array-specific scaling factor. This scaling factor was calculated by dividing a “reference 75th Percentile value” (set as 1500 for the whole series) by the 75th Percentile value of the particular Microarray (“Array i” in the formula shown below). Accordingly, normalized gPS values for all samples (microarray data sets) were calculated by the following formula: normalized gPSArray i = gPSArray i x (1500 / 75th PercentileArray i) A lower intensity threshold was defined as 1% of the reference 75th Percentile value (= 15). All of those normalized gPS values that fell below this intensity border, were substituted by the respective surrogate value of 15.
|
| |
|
| Submission date |
Sep 30, 2013 |
| Last update date |
Jun 23, 2023 |
| Contact name |
Mathias W. Hornef |
| E-mail(s) |
mhornef@ukaachen.de
|
| Phone |
+491702210495
|
| Organization name |
RWTH Áachen University
|
| Department |
Medical Microbiology
|
| Street address |
Steppenbergallee 203
|
| City |
Aachen |
| ZIP/Postal code |
52074 |
| Country |
Germany |
| |
|
| Platform ID |
GPL4134 |
| Series (1) |
| GSE51269 |
Homeostatic intestinal epithelial gene expression |
|
