|
| Status |
Public on Sep 17, 2013 |
| Title |
muscle Smad4KO 2 denervated 14 days |
| Sample type |
RNA |
| |
|
| Source name |
Gastrocnemius muscle denervated 14 days, Smad4 KO mouse
|
| Organism |
Mus musculus |
| Characteristics |
tissue: whole Gastrocnemius
gender: adult male
strain: CD1
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared from Gastrocnemius muscles using TRIzol (Life Technologies) following the manufacturer's recommendations.Then a clean-up step and an on-column DNase I treatment were performed using Rneasy Fibrous Tissue Mini Kit (QIAGEN). RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 1 ug RNA using the One-Color Microarray-Based Gene Expression Analysis (Quick Amp Labeling) (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 >9.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 μl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 μl of 2x GEx Hybridization buffer HI-RPM was added to the fragmentation mixture and hybridized to Agilent Whole Mouse Genome Oligo 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 at 37°C with GE Wash buffer 2 (Agilent), then dried.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5μm, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression after 14 days of denervation
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.5.1.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 014868_D_20070820) to obtain background subtracted and spatially detrended Processed Signal intensities.
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| |
|
| Submission date |
Aug 13, 2013 |
| Last update date |
Sep 17, 2013 |
| Contact name |
Marco Sandri |
| Organization name |
VIMM (Venetian Institute of Molecular Biology)
|
| Street address |
via Orus,2
|
| City |
Padua |
| ZIP/Postal code |
35129 |
| Country |
Italy |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE49826 |
Gene expression profiling on innervated and denervated muscles of Smad4 KO and control mice. |
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