|
| Status |
Public on Apr 01, 2022 |
| Title |
WT heart 6 |
| Sample type |
RNA |
| |
|
| Source name |
left ventricle from 45wk old WT rats
|
| Organism |
Rattus norvegicus |
| Characteristics |
genotype/variation: WT
tissue: left ventricle
gender: male
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Tri-Reagent. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was assessed using an Agilent 2100 Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 100 ng RNA using the Low Input QuickAmp Labeling Kit One-Color (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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| |
|
| Hybridization protocol |
600 ng of Cy3-labeled cRNA (specific activity >6.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 µl of 2x Agilent Hi-RPM hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Mouse Genome Microarrays (G4122F) 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). Next, microarrays were washed 30 seconds in acetonitrile and 30 seconds in Stabilization and Drying Solution.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent G2565CA Microarray Scanner using one color scan setting for 8x60k array slides.
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software (Agilent) using default parameters (protocol GE1-107_Sep09 and Grid: 028279_D_20131202) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. Data were normalized using R statistical software. Raw values of all probes on all arrays were normalized against a virtual median chip (median raw intensity per row) using a locally weighted scattered plot smoother analysis (LOWESS). Values for replicate probes were averaged. The data were filtered to 20,000 probes to remove probes with low intensity values.
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| |
|
| Submission date |
Apr 24, 2020 |
| Last update date |
Apr 01, 2022 |
| Contact name |
Marja Steenman |
| E-mail(s) |
marja.steenman@inserm.fr
|
| Organization name |
L'institut du thorax
|
| Street address |
8 quai Moncousu
|
| City |
Nantes |
| ZIP/Postal code |
44007 |
| Country |
France |
| |
|
| Platform ID |
GPL15084 |
| Series (1) |
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