|
| Status |
Public on Nov 26, 2007 |
| Title |
Testicle tissue, Porcine, D_004107 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Testicle tissue, Porcine, High androstenone, biomat-id 1617
|
| Organism |
Sus scrofa |
| Characteristics |
Breed: Duroc, Gender: Male, Tissue: Testicle, High androstenone, biomat-id 1617
|
| Biomaterial provider |
na
|
| Treatment protocol |
na
|
| Growth protocol |
na
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total-RNA extractions with DNase treatment using RNeasy Midi Kit from Qiagen
|
| Label |
Alexa-594
|
| Label protocol |
20 µg total RNA was labelled by using the Superscript Indirect cDNA Labeling System (Invitrogen) in combination with ARES cDNA labeling kits (Molecular Probes/Invitrogen) following the enclosed protocols. Spike-in RNA from the Lucidea Universal ScoreCard (Amersham Biosciences) was added to the cDNA reactions. ""Green"" spike-in RNA was added to the samples labeled with Alexa-594 and ""red"" spike-in RNA was added to the samples labeled with Alexa-488.
|
| |
|
| Channel 2 |
| Source name |
Testicle tissue, Porcine, Low androstenone, biomat-id 5432
|
| Organism |
Sus scrofa |
| Characteristics |
Breed: Duroc, Gender: Male, Tissue: Testicle, Low androstenone, biomat-id 5432
|
| Biomaterial provider |
na
|
| Treatment protocol |
na
|
| Growth protocol |
na
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total-RNA extractions with DNase treatment using RNeasy Midi Kit from Qiagen
|
| Label |
Alexa-488
|
| Label protocol |
20 µg total RNA was labelled by using the Superscript Indirect cDNA Labeling System (Invitrogen) in combination with ARES cDNA labeling kits (Molecular Probes/Invitrogen) following the enclosed protocols. Spike-in RNA from the Lucidea Universal ScoreCard (Amersham Biosciences) was added to the cDNA reactions. ""Green"" spike-in RNA was added to the samples labeled with Alexa-594 and ""red"" spike-in RNA was added to the samples labeled with Alexa-488.
|
| |
|
| |
| Hybridization protocol |
Two labeled RNA extract batches, corresponding to two independent RNA extractions, were hybridized to cDNA microarrays on a Discovery XT hybridization station from Ventana. The slides were hybridized in a Discovery XT hybridization station (Ventana Discovery Systems, Tucson, AZ, USA). Transfer Chip Prep-2 from 4 ºC to room temperature 1 hour before use. Prepare ChipSpread by mixing equal volumes of ChipSpread A (20 mg/mL BSA, 4x SSC, 0.5 mg/mL sodium azide) and B (formamide; 2 mg/mL SDS) and incubate at room temperature for 1 hour before use. A total of 2.5 mL is needed per slide. Print labels, trim them and place them on the slides. Mix the Chip Map reagents (Chip Prep-1, -2 and - 3) by inversion, remove the cap and place the reagents in the Discovery. Place the slides in the machine and initiate the run. Cover slide with 2.5 mL ChipSpread when the message appears (after few minutes). The machine now runs for app. 1.5 hours to pre-hybridize the slides. Heat a waterbath to 90°C or use a PCR machine. Mix the Chiphybe80, add 200 µL to the sample (<20 µL) and mix carefully. Heat the sample mixture at 90°C for 3 minutes and mix carefully by pipetting. Press ""button"" on the machine which then prepares the slides for hybridization. When the message appears apply the samples onto the slides and press ""button"" and the machine hybridizes at 48 ºC for 6 hours. Wipe oil from backside of slides using a clean-room napkin and place slides in the slide-holder from the High Throughput Wash Station (Telechem, cat.no. HTW) placed in a mTub filled with RiboWash. If processing more than 20 slides, place equal number of slides in two slide-holders and continue in parallel. Transfer the slide-holder to a HTW filled with RiboWash and wash for 2 min with magnetic stirring at 700 rpm. Refill the HTW with RiboWash and repeat the wash. Dip the slide-holder in 2x SSC filled in a mTub (200 mL 20x SSC, Elga H2O + 1800 mL water). Transfer the slide-holder to a HTW filled with 2x SSC and wash for 2 min with magnetic stirring at 700 rpm. Refill the HTW with 2x SSC and repeat the wash. Dip the slide-holder 10 times in 0.1x SSC filled in a mTub (5 mL 20x SSC, Elga H2O + 995 mL water) and leave the holder submerged in 0.1x SSC. Transfer the slides to a mBox slide holder placed in a mTub filled with Elga H2O. Dry arrays by centrifugation (at 300 x g for 4 min placed in a mBox)
|
| Scan protocol |
Scanner: ScanArray Express HT system (Perkin Elmer), 5 µm resolution, 100 % laser power and PMT adjusted individually for each channel. Image analysis software: ScanArray Express (version 3.0, Perkin Elmer) using the histogram method with default settings.
|
| Description |
High and low androstenone testicle tissue samples were prepared from Duroc boars slaughtered at approximately 100kg
|
| Data processing |
The edwards method of the limma Bioconductor package was used for background correction, and the printtip loess method was used for normalizing the expression log-ratios. The empirical Bayes statistics of limma was used to assess differential expression.
|
| |
|
| Submission date |
Mar 30, 2007 |
| Last update date |
Nov 26, 2007 |
| Contact name |
Jakob Hedegaard |
| E-mail(s) |
Jakob.Hedegaard@ki.au.dk
|
| Phone |
(+45)89991363
|
| Organization name |
Aarhus University, Faculty of Agricultural Sciences
|
| Department |
Department of Genetics and Biotechnology
|
| Lab |
Molecular Genetics and System Biology
|
| Street address |
PO-box 50
|
| City |
Tjele |
| ZIP/Postal code |
DK-8830 |
| Country |
Denmark |
| |
|
| Platform ID |
GPL3608 |
| Series (1) |
| GSE7409 |
Gene expression profiles in testis of pigs with extreme high and low levels of androstenone. |
|
