|
| Status |
Public on Mar 31, 2006 |
| Title |
212_EtOH_3_Cy3_vs_EtOH_4_Cy5 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source Name |
LEX_EtOH_4_Cy5
|
| Organism(s) |
Drosophila melanogaster |
| Characteristics |
Strain: Oregon-R
Sex: Female
Age: 4-10 days (after adult emergence)
Temperature:23 degrees Celsius
Light:Dark: 16:8
Relative Humidity: 60%
Label type: Normal
|
| Treatment protocol |
Treatment: 2 mL of 99% ethanol, application was topical using a Potter's tower and the time elapsed between application and sampling was 6 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from approximately 240 adult female Drosophila was isolated using the acid guanidium thiocyanate-phenol-choloroform extraction method (Chomczynski and Sacchi, 1987). Samples were homogenized in 200 μL of solution D using a Kontes pestle and further homogenized with 3 short sonicator pulses after adding an additional 800 μL of solution D. Insoluble material was removed from samples by centrifuging at 16 000 x g for 3 minutes and transferring the supernatant to new microtubes. Three washes with cold 80% EtOH were performed to ensure the removal of red coloured eye pigments and organic contaminants from the RNA pellet. RNA samples with (A260/A280) values between 1.8 and 2.0 were considered suitable for microarray analysis.
|
| Label |
Cy5
|
| |
|
| Channel 2 |
| Source Name |
LEX_EtOH_3_Cy3
|
| Organism(s) |
Drosophila melanogaster |
| Characteristics |
Strain: Oregon-R
Sex: Female
Age: 4-10 days (after adult emergence)
Temperature:23 degrees Celsius
Light:Dark: 16:8
Relative Humidity: 60%
Label type: Normal
|
| Treatment protocol |
Treatment: 2 mL of 99% ethanol, application was topical using a Potter's tower and the time elapsed between application and sampling was 6 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from approximately 240 adult female Drosophila was isolated using the acid guanidium thiocyanate-phenol-choloroform extraction method (Chomczynski and Sacchi, 1987). Samples were homogenized in 200 μL of solution D using a Kontes pestle and further homogenized with 3 short sonicator pulses after adding an additional 800 μL of solution D. Insoluble material was removed from samples by centrifuging at 16 000 x g for 3 minutes and transferring the supernatant to new microtubes. Three washes with cold 80% EtOH were performed to ensure the removal of red coloured eye pigments and organic contaminants from the RNA pellet. RNA samples with (A260/A280) values between 1.8 and 2.0 were considered suitable for microarray analysis.
|
| Label |
Cy3
|
| Label protocol |
80 μg of total RNA was labeled using the direct labeling protocol as specified in Neal et al. (2003), Genome 46: 879-892.
|
| |
|
| |
| Hybridization protocol |
Hybridization protocol as specified in Neal et al. (2003), Genome 46: 879-892. 80 μg of total RNA from the control and treated samples were hybridized.
|
| Scan protocol |
Scan protocol using the ScanArray 4000 XL (GSI Lumonics/Packard Biochips) as specified in Neal et al. (2003), Genome 46: 879-892.
|
| Description |
This hybridization compared the gene expression profiles after 6 hours of two separate pools of Drosophila which received identical treatments. All flies were treated topically with 99% ethanol.
|
| Data processing |
Quantarray Microarray Analysis Software version 3.0 (Packard BioScience, copyright 2001) was used to quantify image data. Spot and background intensity were measured using the adaptive circle quantification method. Gene TrafficTM Duo, version 2.8 (Iobion Informatics, copyright 2002) was used for further analysis. Data normalization was performed on background subtracted spots on a subgrid basis using the Locally Weighted Scatter Plot Smoother (LOWESS) algorithm with a smoothing factor of 20. Spots with intensity values less than 100 units and spots with intensity values below the average background intensity value and/or below the local background intensity value were excluded from normalization and analysis. Data was filtered so as to exclude all genes with less than two-thirds of spots being usable as defined by quality filters and by a mean differential expression ratio with a coefficient of variance higher than 100%.
|
| |
|
| Submission date |
Jul 30, 2005 |
| Contact name |
Helen Rose Jensen |
| E-mail(s) |
Helen.Jensen@alumni.uottawa.ca
|
| Organization name |
University of Ottawa
|
| Department |
Ottawa-Carleton Institute of Biology
|
| Lab |
Center for Advanced Research in Environmental Genomics
|
| Street address |
30 Marie-Curie street
|
| City |
Ottawa |
| State/province |
Ontario |
| ZIP/Postal code |
K1N 6N5 |
| Country |
Canada |
| |
|
| Platform ID |
GPL1473 |
| Series (1) |
| GSE3036 |
Like-like hybridization to test 7K3 platform variability |
|
| Data table header descriptions |
| ID_REF |
|
| CH1_RAW |
Raw fluorescence intensity - Cy5 |
| CH1_BKD_RAW |
Raw background fluorescence intensity - Cy5 |
| CH1_AREA |
Spot area - Cy5 |
| Ch1_Signal_Noise_Ratio |
Signal to noise ratio - Cy5 |
| CH2_RAW |
Raw fluorescence intensity - Cy3 |
| CH2_BKD_RAW |
Raw background fluorescence intensity - Cy3 |
| CH2_AREA |
Spot area - Cy3 |
| Ch2_Signal_Noise_Ratio |
signal to noise ratio -Cy3 |
| Flag |
Flagged data points: A=Manually flagged by user within GeneTraffic; B=Flagged when uploaded; C=Control spot (defined in array layout); R=Reference spot (defined in array layout); F=LEX.E to local background intensity ratio less than specified; G=LEX.R to local background intensity ratio less than N; I=LEX.E intensity less than N times hybridization average background; J=LEX.R intensity less than N times hybridization average background; K=LEX.E intensity less than N; L=LEX.R intensity less than N; H=Housekeeping spot (defined in array layout). Spots flagged as: A,B,F,G,I,J,K or L are exluded from analysis. Spots flagged as C,H or R are not excluded from analysis. |
| Ratio |
Ratio of Ch2_raw to Ch1_raw |
| VALUE |
The log2-transformed ratio of the Lowess-normalized fluorescence values (Ch2/Ch1) exported from GeneTraffic |
