|
| Status |
Public on Sep 26, 2008 |
| Title |
trxE3/trxB11_larvae_(Replicate2) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
w1118;+;trxE3/trxB11 isogenized larvae (Biological Replicate 1)
|
| Organism |
Drosophila melanogaster |
| Characteristics |
Strain: w1118;+;trxE3/trxB11 isogenized from wt strain.
Stage: third instar larvae (bromophenol blue staged)
Tissue: whole larvae
|
| Growth protocol |
Flies were kept on standard media with 0.025% bromophenol blue at 21ºC.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Larvae were squashed in PBS and total RNA extraction was performed with RNeasy Protect Mini Kit (QIAGEN Inc.). Quality was assessed 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).
|
| |
|
| Channel 2 |
| Source name |
w1118;+;+ isogenic wt larvae (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: whole larvae
|
| Growth protocol |
Flies were kept on standard media with 0.025% bromophenol blue at 21ºC.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Larvae were squashed in PBS and total RNA extraction was performed with RNeasy Protect Mini Kit (QIAGEN Inc.). Quality was assessed using the Eukaryote Total RNA Nano Assay on a 2100 Bioanalyzer (Agilent Technologies).
|
| 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).
|
| |
|
| |
| 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 |
Total RNA from w1118;+;+ larvae was pooled and used as a common reference in four microarrays against w1118;+;trxE3/trxB11 total RNA coming from two different extractions to take biological differences into account. Two amplifications from w1118;+;+ and one from each w1118;+;trxE3/trxB11 replicate were performed with the Amino-Allyl Messageamp II aRNA Amplification Kit (Ambion, Inc) to obtain amplified RNA (aRNA). The arrays from each replicate pair were hybridized with the same amplified RNA from sample and common reference 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 |
Aug 10, 2007 |
| Last update date |
Sep 26, 2008 |
| Contact name |
Sergi Beltran |
| Organization name |
Universitat de Barcelona
|
| Department |
Serveis Cientificotècnics
|
| Lab |
Unitat de Bioinformàtica
|
| Street address |
Baldiri Reixac 10
|
| City |
Barcelona |
| ZIP/Postal code |
08028 |
| Country |
Spain |
| |
|
| Platform ID |
GPL3797 |
| Series (2) |
| GSE8748 |
Chromosomal clustering of genes in trithorax (trx) mutant larvae |
| GSE8783 |
trx regulatory gene network in D. melanogaster |
|
| Data table header descriptions |
| ID_REF |
|
| VALUE |
Log2ratio of mutant/wt obtained by normalizing with Bioconductor (see Data Processing). 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 spot which is a negative control or did not pass quality filters. 0.02 indicates a spike-in control which passed quality filters. |
