|
| Status |
Public on Dec 24, 2011 |
| Title |
MP.3.R4 WT.healthy |
| Sample type |
RNA |
| |
|
| Source name |
diabetic ischemic limbs
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
genotype/variation: p75NTR+/+ (WT)
disease state: healthy
tissue: skin
gender: male
|
| Treatment protocol |
Three day old wounds from diabetic and healthy, wildtype and p75KO mice were sampled.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNeasy AllPrep column extraction (Qiagen) using the manufacturers protocols
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 50 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. The Sample data table values are quantile-normalised background-substracted log2-values (using R/Bioconductor package limma).
|
| |
|
| Submission date |
Dec 22, 2011 |
| Last update date |
Dec 24, 2011 |
| Contact name |
Klemens Vierlinger |
| E-mail(s) |
klemens.vierlinger@ait.ac.at
|
| Organization name |
AIT
|
| Department |
HMD
|
| Street address |
Giefinggasse 4
|
| City |
Vienna |
| ZIP/Postal code |
1210 |
| Country |
Austria |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE34675 |
Gene Expression from diabetic ischemic limbs in WT and p75KO mice |
|
