|
| Status |
Public on Dec 01, 2011 |
| Title |
WA_221_No_999_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
skeletal muscle, WA, 221, No HGP, replicate 3
|
| Organism |
Bos indicus |
| Characteristics |
tissue: Biopsied Longissimus dorsi
site: Western Australia WA
genotype: 221
treatment: none
|
| Treatment protocol |
HGP treatment group received Revalor-H (200mg Trenbolone acetate and 20mg estradiol) implants 2 months prior to collection of samples. Needle biopsy samples from Longissimus dorsi muscle were collected with animals under local anaesthetic.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from muscle biopsy samples using TRIZOL (Invitrogen); with the aqueous phase being processed through Rneasy Mini Kit columns (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 0.5 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 |
1.5 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, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) 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), 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 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression after no HGP treatment in WA with 221 genotype
The tenderness genotypes are based on alleles for the genes Calpastatin, Calpain 3 and Calpain 1; where the numbers indicate tender/favoured = 2, intermediate = 1 and tough/unfavoured = 0.
For example 221 means that an animal has favoured genotype for Calpastatin, Calpain 3 and the intermediate genotype for Calpain 1.
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
Oct 28, 2010 |
| Last update date |
Dec 01, 2011 |
| Contact name |
Sean McWilliam |
| E-mail(s) |
sean.mcwilliam@csiro.au
|
| Organization name |
CSIRO
|
| Department |
Agriculture and Food
|
| Street address |
306 Carmody Road
|
| City |
St Lucia |
| State/province |
Queensland |
| ZIP/Postal code |
4067 |
| Country |
Australia |
| |
|
| Platform ID |
GPL9712 |
| Series (1) |
| GSE25005 |
Gene expression study of bovine skeletal muscle |
|
