|
| Status |
Public on Jan 18, 2011 |
| Title |
dsap18 embryos (Biological Replicate1, Dye-swap 2) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
w1118;dsap18^117/Df(3R)sbd^45 (Mutant)
|
| Organism |
Drosophila melanogaster |
| Characteristics |
strain: dsap18^117 corresponds to a null mutation generated by P-element mobilization from EP(3)3462. Df(3R)sbd45, which uncovers dsap18 , was obtained from the Bloomington Stock Center.
stage: embryo
age: 0-24h old
tissue: whole embryos
|
| Growth protocol |
Flies were kept on standard media at 25ºC.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Embryos were dechorionated and total RNA extraction was performed with RNeasy 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 dsap18-HA;dsap18^117/Df(3R)sbd^45 (Control)
|
| Organism |
Drosophila melanogaster |
| Characteristics |
strain: Rescue (control) flies carry a dsap18-HA transgene in chrX.
stage: embryo
age: 0-24h old
tissue: whole embryos
|
| Growth protocol |
Flies were kept on standard media at 25ºC.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Embryos were dechorionated and total RNA extraction was performed with RNeasy 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 |
A total of four microarrays were hybridized in biological replicate pairs, such that the total RNA from dsap18 mutant animals (dsap18^117/Df(3R)sbd^45 0-24h staged embryos) and control animals (rescued dsap18^117/Df(3R)sbd^45 0-24h embryos containing a dsap18-HA transgene) used as 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). Quality of spots was assesed by spot size, foreground versus background signals, saturation and coincidence between differently calculated ratio measures and R2 of regression ratio.
|
| |
|
| Submission date |
Jul 13, 2009 |
| Last update date |
Jan 18, 2011 |
| 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 (1) |
| GSE17066 |
Whole genome expression profile of dsap18 mutant Drosophila melanogaster embryos |
|
| Data table header descriptions |
| ID_REF |
|
| VALUE |
same as UNF_VALUE but with flagged values removed: Log2ratio of mutant/control obtained by normalizing with OLIN in Bioconductor (see Data Processing) |
| 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). |
