|
| Status |
Public on Dec 31, 2010 |
| Title |
Larva, 19˚C, Ubx, Biol. Repl. 2 |
| Sample type |
RNA |
| |
|
| Source name |
Third instar larval wing discs from the experimental Ubx line grown at 19˚C
|
| Organism |
Drosophila melanogaster |
| Characteristics |
developmental stage: Wandering third instar larvae (staged by gut colour intensity)
growth temperature: Exclusively at 19˚C
genotype: w; UAS-UbxIa / + ; nabGal4NP3537 / Tub-Gal80ts
|
| Treatment protocol |
About 15-20 animals per sample were transferred on a silicone rubber plate for dissection in 1xPBS within 20min. Dissected wings were transferred with a pipette tip (pre-coated with BSA) to a clean drop of 1xPBS. Intact wings were transferred to a 1.5ml RNAse-free tube, lysed in 300µl Trizol reagent (Invitrogen), flash frozen in liquid nitrogen and stored at -80 ºC.
|
| Growth protocol |
For sample collection, animals were grown on standard cornmeal-agar medium, strictly in uncrowded conditions. We used three different temperature-controlled incubators (LMS) at 19˚C, 25˚C and 29˚C. In all temperature shift experiments, crosses were kept at 19˚C and were shifted at the appropriate stage to 29˚C via a one-hour-incubation at 25˚C to minimize any heat-shock responses.
Specific protocol: Flies were reared on standard medium supplemented with bromophenol blue (1.5g/l) at 19˚C. Wandering third instar larvae were staged by selecting for a medium blue gut colour intensity.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
From each sample, total RNA was extracted with Trizol (Invitrogen) followed by purification with the RNeasy Micro Kit (Qiagen), both performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
For each sample, biotinylated cRNA was prepared from 2µg total RNA according to standard Affymetrix protocols.
|
| |
|
| Hybridization protocol |
10µg of fragmented cRNA were hybridized in 160μl Hyb buffer for 16hrs at 45˚C on the GeneChip Drosophila Genome 2.0 Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400 using standard Affymetrix protocols.
|
| Scan protocol |
GeneChips were scanned using a GeneChip Scanner 3000, and array images were analyzed with the GCOSv1.4 software.
|
| Description |
Experimental larval wing discs developed at the permissive low temperature
|
| Data processing |
Gene expression data were analyzed with R/Bioconductor (packages: affy, simpleaffy, genefilter and RankProd). We pre-processed and normalized data by RMA, we filtered out the Affymetrix quality control probe-sets and genes that are not expressed above 5 log2 units, and identified the up- and down-regulated genes using the Rank Products non-parametric method. The False Discovery Rate for each Affymetrix probe-set was computed from 1000 permutations, and a significance cut-off of 0.05 was selected.
|
| |
|
| Submission date |
Jun 15, 2010 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Anastasios Tassos Pavlopoulos |
| E-mail(s) |
[email protected]
|
| Phone |
0044 (0)1223 331773
|
| Fax |
0044 (0)1223 336679
|
| URL |
http://www.zoo.cam.ac.uk/zoostaff/mml/akam/members/tassos.htm
|
| Organization name |
University of Cambridge
|
| Department |
Department of Zoology
|
| Lab |
Laboratory for Development and Evolution (Akam group)
|
| Street address |
Downing Street
|
| City |
Cambridge |
| ZIP/Postal code |
CB2 3EJ |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL1322 |
| Series (1) |
| GSE22354 |
Drosophila expression data from larval, prepupal and pupal wings to identify targets of Hox protein Ultrabithorax (Ubx) |
|
| Relations |
| Reanalyzed by |
GSE119084 |
