|
| Status |
Public on Apr 22, 2010 |
| Title |
Piau 515 vs Piau 386 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Skeletal muscle Piau 515
|
| Organism |
Sus scrofa |
| Characteristics |
breed type: Piau
tissue type: Skeletal muscle – Longissimus dorsi
age: 70 days of gestation
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from 30 mg of tissue was extracted using RNeasy Mini kit with on-column RNase-free DNase digestion (Qiagen, Valencia, CA) according to the manufacturer’s instructions. RNA concentration and quality were determined with an RNA 6000 Pico LabChip® kit using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Palo Alto, CA, USA).
|
| Label |
Cy3
|
| Label protocol |
One µg of total RNA was reverse transcribed with a T7 oligo(dT) primer using the Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion Inc., Austin, TX, USA) according to the manufacturer’s instructions. Following first-strand and second-strand synthesis and purification, the cDNAs were in vitro transcribed to synthesize multiple copies of amino allyl-modified aRNAs. After purification, aRNAs were labeled with N-hydroxysuccinate (NHS) ester Cy3 (GE Healthcare, UK).
|
| |
|
| Channel 2 |
| Source name |
Skeletal muscle Piau 386
|
| Organism |
Sus scrofa |
| Characteristics |
breed type: Piau
tissue type: Skeletal muscle – Longissimus dorsi
age: 40 days of gestation
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from 30 mg of tissue was extracted using RNeasy Mini kit with on-column RNase-free DNase digestion (Qiagen, Valencia, CA) according to the manufacturer’s instructions. RNA concentration and quality were determined with an RNA 6000 Pico LabChip® kit using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Palo Alto, CA, USA).
|
| Label |
Cy5
|
| Label protocol |
One µg of total RNA was reverse transcribed with a T7 oligo(dT) primer using the Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion Inc., Austin, TX, USA) according to the manufacturer’s instructions. Following first-strand and second-strand synthesis and purification, the cDNAs were in vitro transcribed to synthesize multiple copies of amino allyl-modified aRNAs. After purification, aRNAs were labeled with N-hydroxysuccinate (NHS) ester Cy5 (GE Healthcare, UK).
|
| |
|
| |
| Hybridization protocol |
Labeled aRNAs were purified and combined with 65 µl of Slide Hyb #1 solution (Ambion, Inc.) and denatured at 70C for 5 min. Hybridizations were performed in sealed hybridization cassettes (ArrayIt, TeleChem International, Inc., Sunnyvale, CA, USA) for 18 h at a humid 54C. Following hybridization, slides were washed in 2X SSC/0.5% SDS and 0.1X SSC/0.1% SDS solutions for 10 min each. The slides were rinsed in a 0.1X SSC solution and nuclease-free water and dried by centrifugation.
|
| Scan protocol |
Fluorescent images were detected by a GenePix 4000B scanner (Molecular Devices, Sunnyvale, CA, USA) and fluorescence intensity data were collected using GenePix software (Molecular Devices) after spot alignment. Median intensity values for each dye channel were stored as comma-separated values data files.
|
| Description |
The tissue samples compared on this array are part of an experiment to study muscle development at 40 and 70 days of gestation in a Brazilian local pig breed and crossbred pigs from the US using the swine protein-annotated oligonucleotide microarray.
|
| Data processing |
Loess normalized log-ratio of Cy5 versus Cy3 intensities. No background correction.
|
| |
|
| Submission date |
Apr 20, 2010 |
| Last update date |
Apr 21, 2010 |
| Contact name |
Catherine W. Ernst |
| E-mail(s) |
[email protected]
|
| Phone |
517-432-1941
|
| Fax |
517-432-0753
|
| URL |
http://www.msu.edu/~ernstc/
|
| Organization name |
Michigan State University
|
| Department |
Animal Science
|
| Lab |
Molecular Genetics
|
| Street address |
3330 Anthony Hall
|
| City |
East Lansing |
| State/province |
MI |
| ZIP/Postal code |
48824 |
| Country |
USA |
| |
|
| Platform ID |
GPL7435 |
| Series (1) |
| GSE21412 |
Transcriptional profiling during fetal skeletal muscle development of Piau and Yorkshire-Landrace crossbred pigs |
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