|
| Status |
Public on Jan 10, 2013 |
| Title |
sevd2_eye_disc_Rep4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
eye_discs_sevd2_(EXTRACTION 2)
|
| Organism |
Drosophila melanogaster |
| Characteristics |
strain: sevd2;+;+
Stage: third instar larvae (bromophenol blue staged)
tissue: eye imaginal discs
genetic background: w1118
|
| Treatment protocol |
none
|
| Growth protocol |
All Drosophila strains and crosses were kept on standard media with 0.025% bromophenol blue at 25ºC.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Eye imaginal discs were dissected in PBS and kept in RNAlater prior to extraction with RNeasy Protect Mini Kit (QIAGEN Inc.). Quality was assessed in all samples using the Eukaryote Total RNA Nano Assay on a 2100 Bioanalyzer (Agilent Technologies). Total RNA from w1118;+;+ eye imaginal discs was pooled and used as a common reference against sevd2 and sevS11
|
| Label |
Cy3
|
| Label protocol |
1.5ug total RNA were amplified and indirectly labelled with Amino-Allyl Messageamp II aRNA Amplification Kit (Ambion, Inc). Fragmentation was done with RNA Fragmentation Reagents #8740(Ambion, Inc).
|
| |
|
| Channel 2 |
| Source name |
eye_discs_wt_(POOL)
|
| Organism |
Drosophila melanogaster |
| Characteristics |
strain: w1118;+;+ isogenic wt strain (Ryder, E., 2004; Genetics 167, 797-813).
Stage: third instar larvae (bromophenol blue staged)
tissue: eye imaginal discs
genetic background: w1118
|
| Treatment protocol |
none
|
| Growth protocol |
All Drosophila strains and crosses were kept on standard media with 0.025% bromophenol blue at 25ºC.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Eye imaginal discs were dissected in PBS and kept in RNAlater prior to extraction with RNeasy Protect Mini Kit (QIAGEN Inc.). Quality was assessed in all samples using the Eukaryote Total RNA Nano Assay on a 2100 Bioanalyzer (Agilent Technologies).
|
| Label |
Cy5
|
| Label protocol |
1.5ug total RNA were amplified and indirectly labelled with Amino-Allyl Messageamp II aRNA Amplification Kit (Ambion, Inc). Fragmentation was done with RNA Fragmentation Reagents #8740(Ambion, Inc).
|
| |
|
| |
| Hybridization protocol |
Samples were diluted in 2X Hybridization Buffer #5185-5973(Agilent Technologies) and hybridization was carried out at 60ºC for 18 hours with a G2534A SureHyb Chamber in a G2545A Hybridization Oven (Agilent Technologies).
|
| Scan protocol |
GenePix Results (GPR) data files were obtained for each microarray with an Axon 4000B scanner and GenePix Pro 6 (Axon Instruments, Inc).
|
| Description |
Description Total RNA from w1118;+;+ wing imaginal discs was pooled and used as a common reference against sevd2 and sevS11. Four microarrays were hybridized for sevd2 experiment in biological replicate pairs, such that the total RNA from the sample used as a starting material came from different extractions. Both arrays from each pair were hybridized with the same amplified RNA from sample and common reference (obtained using the Amino-Allyl Messageamp II aRNA Amplification Kit from Ambion, Inc) but with dyes (Cy3 and Cy5 from Amersham, Inc) swapped to take dye-bias into account.
|
| Data processing |
GPR files were analyzed with Limma package from BioConductor (Gentleman et al., 2004; Genome Biol 5, R80; Smyth, 2004; Statistical Applications in Genetics and Molecular Biology 3, Article 3) using the same criteria. Data was background corrected with the "normexp" method and normalized with OLIN (Futschik and Crompton, 2005; Bioinformatics 21, 1724-1726). Spots not fulfilling the quality thresholds (based on spot size, foreground versus background signals, saturation, coincidence between differently calculated ratio measures and R2 of regression ratio) were eliminated from further analysis.
|
| |
|
| Submission date |
May 06, 2012 |
| Last update date |
Jan 10, 2013 |
| Contact name |
Natalia Mora |
| E-mail(s) |
n.mora@ub.edu
|
| Phone |
0034 934035875
|
| Organization name |
University of Barcelona
|
| Department |
Genetics
|
| Lab |
genetics development
|
| Street address |
avd. Diagonal 643
|
| City |
barcelona |
| State/province |
barcelona |
| ZIP/Postal code |
08028 |
| Country |
Spain |
| |
|
| Platform ID |
GPL3797 |
| Series (1) |
| GSE37793 |
Transcriptome of sevenless (sev) in the eye imaginal disc. |
|
| Data table header descriptions |
| ID_REF |
|
| VALUE |
Normalized log2ratio mutant/control. null means the spot did not pass quality filters or is a negative or spike-in control. |
| F635_MEDIAN |
Median feature pixel intensity at wavelength 635 nm (Cy5). |
| B635_MEDIAN |
Median feature background intensity at wavelength 635 nm (Cy5). |
| F532_MEDIAN |
Median feature pixel intensity at wavelength 532 nm (Cy3). |
| B532_MEDIAN |
Median feature background intensity at wavelength 532 nm (Cy3). |
| RATIO_OF_MEDIANS_(635/532) |
The ratio of the median intensities of each feature for each wavelength, with the median background subtracted.Not normalized. |
| MEDIAN_OF_RATIOS_(635/532) |
The median of pixel-by-pixel ratios of pixel intensities, with the median background subtracted.Not normalized. |
| RGN_RATIO_(635/532) |
The regression ratio of every pixel in a 2-feature-diameter circle around the center of the feature.Not normalized. |
| RGN_R2_(635/532) |
The coefficient of determination for the current regression value. |
| WEIGHT |
0.01 indicates a negative control or bad quality spot. 0.02 indicates a good quality spike-in control spot. |
| UNF_VALUE |
Log2ratio of mutant/control obtained by normalizing with Bioconductor (see Data Processing). |
