|
| Status |
Public on Mar 07, 2014 |
| Title |
Liver_low forage allowance_purebred_+15 days_replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
Liver, low forage allowance, purebred, +15 days, replicate 2
|
| Organism |
Bos taurus |
| Characteristics |
genotype: AA
tissue: liver
developmental stage: mature cow
herbage allowance: low forage allowance
time: +15 days (early lactation)
|
| Treatment protocol |
Liver biopsies were collected using a 14-gauge biopsy needle (Tru-Core-II Automatic Biopsy Instrument; Angiotech, Lausanne, Switzerland), at -165 and -15 ± 8 (gestation), +15 and +60 ± 3 (early lactation) days ± SD. Samples were immediately frozen in liquid nitrogen and stored at -80°C until analyzed.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRIZOL (Invitrogen, Life Technologies, Carlsbad, CA, USA) followed by precipitation with lithium chloride and DNase-treatment (Applied Biosystems/Ambion, Austin,TX, USA). 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.
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| |
|
| 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 |
Liver gene expression of mature cow in , low forage allowance, purebred, +15 days, replicate 2
3100 +15
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 023647_D_F_20090518) 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 |
Nov 05, 2013 |
| Last update date |
Mar 07, 2014 |
| Contact name |
Mariana Carriquiry |
| E-mail(s) |
mcarriquiry@fagro.edu.uy
|
| Phone |
+59823572344
|
| Organization name |
Facultad de Agronomía, UdelaR
|
| Department |
Department of Animal Sciences and Pastures
|
| Street address |
Ave Garzon 780
|
| City |
Montevideo |
| ZIP/Postal code |
12300 |
| Country |
Uruguay |
| |
|
| Platform ID |
GPL11648 |
| Series (1) |
| GSE52085 |
Liver functional genomics in beef cows on grazing systems |
|
