|
| Status |
Public on Feb 25, 2017 |
| Title |
Group 3, Nox ,19 |
| Sample type |
RNA |
| |
|
| Source name |
lung tissue
|
| Organism |
Mus musculus |
| Characteristics |
age: harvested at P5.5
treatment: normoxia
|
| Treatment protocol |
Pups were randomized on the day of birth and litters were balanced. 4 cages were placed in normoxia whereas 4 cages in hyperoxia. Then, pups of the same age being placed in normoxia or hyperoxia were sacrificed on the same time-point.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mice were euthanized with pentobarbital for early time-points (from P2.5 to P5.5) or with isoflurane for P14.5. Thoracic cavities were opened and lungs were perfused through the heart with PBS until lungs were white colored. Lungs were harvested, placed into tubes and immediately frozen to liquid nitrogen and homogenized with Precellys 24 homogenizer. RNA isolation was performed with the miRNeasy Kit (Qiagen 217004) following the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.1 ug RNA using the One-Color Low Input Quick-Amp Labeling 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 |
0.6 ug of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent-035430 8x60K Mouse miRNA Rel17 Microarray (GPL15547) for 20 hours at 55°C in a rotating 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), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol miRNA_107_Sep09 and Grid: 035430_D_F_20111226 ) to obtain background subtracted and spatially detrended Processed Signal intensities. Features which were not positive and significant, not above background or were population outliers were flagged as “Compromised”. If features were flagged “Compromised” in all samples, than they were excluded.
|
| |
|
| Submission date |
Nov 08, 2016 |
| Last update date |
Feb 27, 2017 |
| Contact name |
Rory Morty |
| Organization name |
MPI für Herz- und Lungenforschung
|
| Department |
W.G. Kerckhoff-Institut
|
| Street address |
Parkstr. 1
|
| City |
Bad Nauheim |
| ZIP/Postal code |
61231 |
| Country |
Germany |
| |
|
| Platform ID |
GPL15547 |
| Series (1) |
| GSE89666 |
Differential microRNA gene expression between normal and aberrant late lung development induced by hyperoxia in mice |
|
