|
| Status |
Public on Aug 22, 2009 |
| Title |
Eyediscs_selectedlines_rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Eyediscs_selectedline_low
|
| Organism |
Teleopsis dalmanni |
| Characteristics |
selected line: low eyespan/bodylength, gen 65
sex: male
tissue: eye antennal imaginal discs
developmental stage: late instar wandering larvae
|
| Biomaterial provider |
Gerald S Wilkinson
|
| Treatment protocol |
Flies had been under selection for 65 generations to decrease eyespan/body length.
|
| Growth protocol |
Larvae were reared at low density in pureed corn media at constant temperature (25 degrees C).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Imaginal disc and brain tissue were dissected from wandering male larvae just after gut purge. Male larvae were identified by genital disc morphology and their eye discs, optic lobes and brains were removed and submerged in RNAlater solution (Ambion) and stored at -20o. Total RNA was subsequently extracted from disc and brain tissue using an RNA isolation kit (Promega). Each biological sample consisted of twenty-five sets of eye discs which were homogenized by grinding with plastic pestles in 175 µl lysis buffer. After treating with DNase I enzyme, RNA was concentrated by lyophilization and the quality of each sample was checked with an Agilent 2100 bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
We used the Ovation amino allyl RNA amplification kit (NuGen) to amplify messenger RNA prior to labeling. This procedure uses reverse transcription to make single-stranded cDNA and then DNA polymerization to make double-stranded (ds) cDNA. The ds cDNA was then amplified using a linear amplification procedure. Samples were labeled using an amino allyl cDNA labeling kit (Ambion)
|
| |
|
| Channel 2 |
| Source name |
Eyediscs_selectedline_high
|
| Organism |
Teleopsis dalmanni |
| Characteristics |
selected line: high eyespan/bodylength, gen 65
sex: male
tissue: eye antennal imaginal discs
developmental stage: late instar wandering larvae
|
| Biomaterial provider |
Gerald S Wilkinson
|
| Treatment protocol |
Flies had been under selection for 65 generations to increase eyespan/body length.
|
| Growth protocol |
Larvae were reared at low density in pureed corn media at constant temperature (25 degrees C).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Imaginal disc and brain tissue were dissected from wandering male larvae just after gut purge. Male larvae were identified by genital disc morphology and their eye discs, optic lobes and brains were removed and submerged in RNAlater solution (Ambion) and stored at -20o. Total RNA was subsequently extracted from disc and brain tissue using an RNA isolation kit (Promega). Each biological sample consisted of twenty-five sets of eye discs which were homogenized by grinding with plastic pestles in 175 µl lysis buffer. After treating with DNase I enzyme, RNA was concentrated by lyophilization and the quality of each sample was checked with an Agilent 2100 bioanalyzer.
|
| Label |
Cy5
|
| Label protocol |
We used the Ovation amino allyl RNA amplification kit (NuGen) to amplify messenger RNA prior to labeling. This procedure uses reverse transcription to make single-stranded cDNA and then DNA polymerization to make double-stranded (ds) cDNA. The ds cDNA was then amplified using a linear amplification procedure. Samples were labeled using an amino allyl cDNA labeling kit (Ambion)
|
| |
|
| |
| Hybridization protocol |
Hybridization followed Agilent protocols and used an Agilent rotator rack and oven.
|
| Scan protocol |
Arrays were scanned with a GenePix 4200A with pixel size set to 5 microns. Temperatute was 23.63.
|
| Description |
To identify genes that influence the length of eyestalks we compared gene expression between lines of flies that have been under artificial sexual selection to increase or decrease male eye span for 65 generations. We compared gene expression between male flies from the high (H2) and low (L2) lines that had deviated furthest from the average control lines. At the time of these experiments the eyespan of H2 male flies was 9.36 ± 0.07 mm and the eyespan of L2 male flies was 7.39 ± 0.07 mm, which represents a difference of 5.7 s.d. between these two lines.
|
| Data processing |
A custom-fitted grid was aligned to the array, and then median signal intensity was calculated for circular features. Data were removed if median intensity/background < 2 or pixel saturation exceeded 70%. Dye bias was corrected using the ratio of medians from all features. Background subtraction was set to Local Feature. Lowess normalization was then applied.
|
| |
|
| Submission date |
Mar 29, 2009 |
| Last update date |
Aug 22, 2009 |
| Contact name |
Gerald S Wilkinson |
| E-mail(s) |
[email protected]
|
| Phone |
301-405-6942
|
| Organization name |
University of Maryland
|
| Department |
Biology
|
| Lab |
Wilkinson
|
| Street address |
Bldg 144, 4094 Campus Dr
|
| City |
College Park |
| State/province |
MD |
| ZIP/Postal code |
20742 |
| Country |
USA |
| |
|
| Platform ID |
GPL8358 |
| Series (1) |
| GSE15444 |
Microarray analysis of stalk-eyed fly lines selected for divergent eyespan |
|
