Strain:Oregon R Gender: female Age: adult 6 days old
Biomaterial provider
Laboratoire de genomique fonctionnelle des insectes
Treatment protocol
Emerging flies were separated by sex and maintained on standard diet for 72h before induction. The flies were then placed on medium containing the inducer at 5mM for atrazine for 72 additional hours.
Growth protocol
All fly stocks were maintained at 21�C on standard corn medium for a 12:12 h dark/light period. Oregon R flies were used as wild type strain.
Extracted molecule
total RNA
Extraction protocol
Standard Operating Procedure Title: RNA Isolation with TRIzol Reagent Protocole from www.invitrogen.com Material safety: When working with TRIzol Reagent use gloves and eye protection. Avoid contact with skin or clothing. Use in a chemical fume hood. Avoid breathing vapour. 1. HOMOGENIZATION Flies were crushed in liquid nitrogen with a mortar. The obtained powder was homogenized in 1 ml of TRIzol Reagent. The sample volume should not exceed 10% of the volume of TRIzol Reagent used for homogenization. Following homogenization, remove insoluble material from the homogenate by centrifugation at 12,000 x g for 10 minutes at 2 to 8�C. The resulting pellet contains extracellular membranes, polysaccharides, and high molecular weight DNA, while the supernatant contains RNA. Transfer the cleared homogenate solution to a fresh tube. 2. PHASE SEPARATION Incubate the homogenized samples for 5 min at 15 to 30�C to permit the complete dissociation of nucleoprotein complexes. Add 0.2 ml of chloroform per 1 ml of TRIzol reagent. Cap sample tubes securely. Shake tubes vigorously by hand for 15 secondes and incubate them at 15 to 30�C for 2 to 3 min. Centrifuge the samples at no more than 12,000 x g for 15 min at 2 to 8�C. Following centrifugation, the mixture separates into a lower red, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. The volume of the aqueous phase is about 60% of the volume of TRIzol Reagent used for homogenization. 3. RNA PRECIPITATION Transfer the aqueous phase to a fresh tube, and save the organic phase if isolation of DNA or protein is desired. Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5 ml of isopropyl alcohol per 1 ml of TRIzol reagent used for the initial homogenization. Incubate samples at 15 to 30�C for 10 min and centrifuge at no more than 12,000 x g for 10 min at 2 to 8�C. The RNA precipitate, often invisible before centrifugation, forms a gel-like pellet on the side and bottom of the tube. 4. RNA WASH Remove the supernatant. Wash the RNA pellet once with 75% ethanol, adding at least 1 ml of 75% ethanol per ml of TRIzol Reagent used for the initial homogenization. Mix the sample by vortexing and centrifuge at no more than 7,500 x g for 5 min at 2 to 8�C. 5. REDISSOLVING THE RNA At the end of the procedure, briefly dry the RNA pellet. It is important not to let the RNA pellet dry completely as this will greatly decrease its solubility. Dissolve RNA in RNA free water.
Label
labelled with Cy3 dCTP from Amersham
Label protocol
Standard Operating Procedure Title: cDNA Labeling Protocol for Total RNA Protocole from www.promega.com 1. For each Cy-dye labeling reaction, assemble the following reagents in a microcentrifuge tube. Keep the reagents on ice, and mix the total RNA and primers as follows: Total RNA 10�g ( unless other specification) Random Primers (3�g/�l) 1�l Oligo(dT) Primer (2�g/�l) 1�l Nuclease-Free Water to a total volume of 20�l 2. Incubate RNA/primer solution at 70�C for 10 minutes ( use the PCR form the lab C314 with PRONTO program), click pause on the PCR machine, take the tubes and place them on ice. 3. While the RNA/primer solution is incubating at 70�C, prepare labeling mix as follows. Perform Cy3 and Cy5 reactions in separate tubes. Component Cy3 Cy5 ChipShot Reverse Transcriptase 5X Reaction Buffer 8�l 8�l MgCl2 (25mM) 4.8�l 4.8�l dNTP mix, Total RNA 2�l 3�l Cy3 dCTP (1mM) 1�l - Cy5 dCTP (1mM) - 1�l ChipShot Reverse Transcriptase 3.2�l 3.2�l Nuclease-Free Water 1�l - final volume 20�l 20�l 4. Add the entire 20�l labeling mix to each tube of RNA/primer solution from step 3, vortex, spin briefly and incubate at room temperature (22-25�C) for 10 minutes ( second step on the PRONTO program of the PCR machine from Lab C314) protected from light. 5. Incubate at 42�C for 2 hours protected from light ( step 3 from PRONTO program of the PCR machine from Lab C314). 6. Add 1.0�l RNase H and 0.35�l RNase Solution to each cDNA-synthesis reaction. Mix gently and incubate at 37�C for 15 minutes ( step 4 from PRONTO program of the PCR machine from Lab C314). Purifying Cy-Labeled cDNA
1. To 40�l of the synthesized, labeled cDNA, add the following components in the order listed: Binding Particles: 18�l Sodium Acetate, 3M (pH 5.2): 4�l Binding Solution: 310�l 2. Vortex gently for 5-10 seconds to mix. 3. Incubate at room temperature for 1 minute. 4. Repeat Steps 2 and 3. 5. Apply entire contents of tube (solution and Binding Particles) to a Spin-X column and cap the tube. 6. Spin at 10,000g for 1 minute. 7. Discard the column flowthrough. 8. Wash column with 500�l of 80% ethanol ( fresly prepared), mix 80%EtOH and binding particles by by up and down with the pipet, cap the tube, and centrifuge at 10,000g for 60 seconds. 9. Discard the column flowt hrough. 10. Repeat Step 8 and 9 twice for a total of 3 washes. 11. Centrifuge column at 10,000g for 1 minute to remove traces of ethanol. 12. Place column in a clean Collection Tube (provided). 13. To elute labeled cDNA, add 60�l of Elution Buffer , mix by up and down the EB with the particles and let the column stand at room temperature for 5 minutes. Centrifuge at 10,000 x gfor 1 minute, discard column. The eluted cDNA can be stored in a light-proof container at 4�C for several weeks.14. Quantitate absorbance at 260, 280, 320 550 and 650nm, and calculate frequency of incorporation. Frequency of incorporation (FOI) is defined as the number of Cy-labeled nucleotides incorporated per 1,000 nucleotides of cDNA. Best results are obtained with cDNAs having an FOI as recommended in Table 2. The FOI can be calculated as follows: FOI = pmol of dye incorporated x 324.5 ng of cDNA Amount of labeled cDNA (ng) = A260 x 37 x total volume (�l) For Cy3: pmol of dye incorporated = [A550 x total volume (�l) ]/0.15 For Cy5: pmol of dye incorporated = [A650 x total volume (�l)]/0.25 Note: These equations were generated using the following constants: Average Molar Mass of dNTP = 324.5; One A260 unit of single-stranded DNA = 37�g/ml; Extinction Coefficient of Cy3 = 150,000M-1cm-1 at 550nm; Extinction Coefficient of Cy5 = 250,000M-1cm-1 at 650nm.
Strain:Oregon R Gender: female Age: adult 6 days old
Biomaterial provider
Laboratoire de genomique fonctionnelle des insectes
Treatment protocol
Emerging flies were separated by sex and maintained on standard diet for 72h before induction. The flies were then placed on medium containing no inducer for 72 additional hours.
Growth protocol
All fly stocks were maintained at 21�C on standard corn medium for a 12:12 h dark/light period. Oregon R flies were used as wild type strain.
Extracted molecule
total RNA
Extraction protocol
Standard Operating Procedure Title: RNA Isolation with TRIzol Reagent Protocole from www.invitrogen.com Material safety: When working with TRIzol Reagent use gloves and eye protection. Avoid contact with skin or clothing. Use in a chemical fume hood. Avoid breathing vapour. 1. HOMOGENIZATION Flies were crushed in liquid nitrogen with a mortar. The obtained powder was homogenized in 1 ml of TRIzol Reagent. The sample volume should not exceed 10% of the volume of TRIzol Reagent used for homogenization. Following homogenization, remove insoluble material from the homogenate by centrifugation at 12,000 x g for 10 minutes at 2 to 8�C. The resulting pellet contains extracellular membranes, polysaccharides, and high molecular weight DNA, while the supernatant contains RNA. Transfer the cleared homogenate solution to a fresh tube. 2. PHASE SEPARATION Incubate the homogenized samples for 5 min at 15 to 30�C to permit the complete dissociation of nucleoprotein complexes. Add 0.2 ml of chloroform per 1 ml of TRIzol reagent. Cap sample tubes securely. Shake tubes vigorously by hand for 15 secondes and incubate them at 15 to 30�C for 2 to 3 min. Centrifuge the samples at no more than 12,000 x g for 15 min at 2 to 8�C. Following centrifugation, the mixture separates into a lower red, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. The volume of the aqueous phase is about 60% of the volume of TRIzol Reagent used for homogenization. 3. RNA PRECIPITATION Transfer the aqueous phase to a fresh tube, and save the organic phase if isolation of DNA or protein is desired. Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5 ml of isopropyl alcohol per 1 ml of TRIzol reagent used for the initial homogenization. Incubate samples at 15 to 30�C for 10 min and centrifuge at no more than 12,000 x g for 10 min at 2 to 8�C. The RNA precipitate, often invisible before centrifugation, forms a gel-like pellet on the side and bottom of the tube. 4. RNA WASH Remove the supernatant. Wash the RNA pellet once with 75% ethanol, adding at least 1 ml of 75% ethanol per ml of TRIzol Reagent used for the initial homogenization. Mix the sample by vortexing and centrifuge at no more than 7,500 x g for 5 min at 2 to 8�C. 5. REDISSOLVING THE RNA At the end of the procedure, briefly dry the RNA pellet. It is important not to let the RNA pellet dry completely as this will greatly decrease its solubility. Dissolve RNA in RNA free water.
Label
labelled with Cy5 dCTP from Amersham
Label protocol
Standard Operating Procedure Title: cDNA Labeling Protocol for Total RNA Protocole from www.promega.com 1. For each Cy-dye labeling reaction, assemble the following reagents in a microcentrifuge tube. Keep the reagents on ice, and mix the total RNA and primers as follows: Total RNA 10�g ( unless other specification) Random Primers (3�g/�l) 1�l Oligo(dT) Primer (2�g/�l) 1�l Nuclease-Free Water to a total volume of 20�l 2. Incubate RNA/primer solution at 70�C for 10 minutes ( use the PCR form the lab C314 with PRONTO program), click pause on the PCR machine, take the tubes and place them on ice. 3. While the RNA/primer solution is incubating at 70�C, prepare labeling mix as follows. Perform Cy3 and Cy5 reactions in separate tubes. Component Cy3 Cy5 ChipShot Reverse Transcriptase 5X Reaction Buffer 8�l 8�l MgCl2 (25mM) 4.8�l 4.8�l dNTP mix, Total RNA 2�l 3�l Cy3 dCTP (1mM) 1�l - Cy5 dCTP (1mM) - 1�l ChipShot Reverse Transcriptase 3.2�l 3.2�l Nuclease-Free Water 1�l - final volume 20�l 20�l 4. Add the entire 20�l labeling mix to each tube of RNA/primer solution from step 3, vortex, spin briefly and incubate at room temperature (22-25�C) for 10 minutes ( second step on the PRONTO program of the PCR machine from Lab C314) protected from light. 5. Incubate at 42�C for 2 hours protected from light ( step 3 from PRONTO program of the PCR machine from Lab C314). 6. Add 1.0�l RNase H and 0.35�l RNase Solution to each cDNA-synthesis reaction. Mix gently and incubate at 37�C for 15 minutes ( step 4 from PRONTO program of the PCR machine from Lab C314). Purifying Cy-Labeled cDNA
1. To 40�l of the synthesized, labeled cDNA, add the following components in the order listed: Binding Particles: 18�l Sodium Acetate, 3M (pH 5.2): 4�l Binding Solution: 310�l 2. Vortex gently for 5-10 seconds to mix. 3. Incubate at room temperature for 1 minute. 4. Repeat Steps 2 and 3. 5. Apply entire contents of tube (solution and Binding Particles) to a Spin-X column and cap the tube. 6. Spin at 10,000g for 1 minute. 7. Discard the column flowthrough. 8. Wash column with 500�l of 80% ethanol ( fresly prepared), mix 80%EtOH and binding particles by by up and down with the pipet, cap the tube, and centrifuge at 10,000g for 60 seconds. 9. Discard the column flowt hrough. 10. Repeat Step 8 and 9 twice for a total of 3 washes. 11. Centrifuge column at 10,000g for 1 minute to remove traces of ethanol. 12. Place column in a clean Collection Tube (provided). 13. To elute labeled cDNA, add 60�l of Elution Buffer , mix by up and down the EB with the particles and let the column stand at room temperature for 5 minutes. Centrifuge at 10,000 x gfor 1 minute, discard column. The eluted cDNA can be stored in a light-proof container at 4�C for several weeks.14. Quantitate absorbance at 260, 280, 320 550 and 650nm, and calculate frequency of incorporation. Frequency of incorporation (FOI) is defined as the number of Cy-labeled nucleotides incorporated per 1,000 nucleotides of cDNA. Best results are obtained with cDNAs having an FOI as recommended in Table 2. The FOI can be calculated as follows: FOI = pmol of dye incorporated x 324.5 ng of cDNA Amount of labeled cDNA (ng) = A260 x 37 x total volume (�l) For Cy3: pmol of dye incorporated = [A550 x total volume (�l) ]/0.15 For Cy5: pmol of dye incorporated = [A650 x total volume (�l)]/0.25 Note: These equations were generated using the following constants: Average Molar Mass of dNTP = 324.5; One A260 unit of single-stranded DNA = 37�g/ml; Extinction Coefficient of Cy3 = 150,000M-1cm-1 at 550nm; Extinction Coefficient of Cy5 = 250,000M-1cm-1 at 650nm.
Hybridization protocol
Standard Operating Procedure Title: Hybridization pronto protocol Protocol from www.promega.com 1. Dry the appropriate amount of each dye-labeled cDNA using a speedvacuum concentrator, then dissolve the cDNA in the required volume of Pronto! Universal Hybridization Solution or in home made hybridization solution ( 1/3 ddH2O+ 2/3 DIGEasy). 2. Incubate the labeled cDNA solution at 95�C for 5 minutes, protecting samples from light. 3. Centrifuge the cDNA at 13,500g for two minutes to collect condensation. Do not place the solution on ice because this will cause precipitation of some of the components. 4. Place array in Corning Hybridization Chamber (Cat.# 2551). Pipet the labeled cDNA gently up and down and then transfer onto the surface of the printed side of the slide. Carefully place the cover glass on the array. Avoid trapping air bubbles between the array and the cover glass. Small air bubbles that do form usually dissipate during hybridization. Assemble the chamber as described in its package insert.
5. Incubate the chamber-array assembly at 42�C for 14-20 hours using a water bath or a hybridization oven. Make sure you place the hybridization chamber horizontally . this can be checked with bubble in the middle of the stuff??
Scan protocol
Slides were scanned on a Genepix 4000b scanner and signal quantification was performed using the Genepix pro 4.1 software (Axon Instruments).
Description
A cDNA microarray was constructed using expressed sequence tags (ESTs) of 1.8 kb average length and gene specific tags of 0.4 kb average length obtained by PCR amplification and plasmid cloning. This Drosophila toxicology microarray contained 319 genes that represent all cytochromes P450, glutathione S-transferases, esterases, other genes of interest including attacin or Ftz-F1 and several housekeeping genes as controls. Each insert was amplified by PCR with flanking universal primers, purified and then spotted using the ChipWriterPro Virtek (BioRad) spotter at three independent locations on Corning UltraGAPS slides (Corning).
Data processing
Data files from Genepix were converted using Express Converter software and normalized in MIDAS (Microarray Data Analysis System), using two publicly available programs from TIGR. Three steps of normalization were performed in MIDAS, (a) a low intensity filter in which signal intensity < 1000 was removed; (b) a global lowess normalization was applied; (c) a flip dye consistency check, the Cy3 and Cy5 labels were swapped between controls versus treatments to account for potential differences in labelling efficiency. For statistical analysis, each array intensity was log-transformed, centred by their average value and reduced by their standard deviation. The resulting data for each gene were analyzed with a type III two-way (i.e., sex x treatment) analysis of variance (ANOVA) taking into account the presence of missing values. A gene was considered as differentially expressed if 50% or more of its variance was explained by the treatment effect with a corresponding P-value lower than 0.035. Data were removed when there was no consistency for a same gene represented on the array by PCR products of different size, as this may have reflected a different degree of specificity.
Xenobiotic response in Drosophila melanogaster: sex dependence of P450 and GST gene induction
Data table header descriptions
ID_REF
X
The X-coordinate of the center of the feature-indicator associated with the feature, where (0,0) is the top left of the image.
Y
The Y-coordinate of the center of the feature-indicator associated with the feature, where (0,0) is the top left of the image.
Dia.
The diameter in micrometer of the feature-indicator.
F635 Median
Median feature pixel intensity at wavelength 635 nm.
F635 Mean
Mean feature pixel intensity at wavelength 635 nm.
F635 SD
The standard deviation of the feature pixel intensity at wavelength 635 nm.
B635 Median
The median background intensity at the wavelength 635 nm.
B635 Mean
The mean background intensity at the wavelength 635 nm.
B635 SD
The standard deviation of the local background intensity at wavelength 635 nm.
% > B635+1SD
The percentage of feature pixels with intensities more than one standard deviation above the background intensity at the wavelength 635 nm.
% > B635+2SD
The percentage of feature pixels with intensities more than two standard deviation above the background intensity at the wavelength 635 nm.
F635 % Sat.
The percentage of the feature pixels at wavelength 635 nm that are saturated.
F532 Median
Median feature pixel intensity at wavelength 532 nm.
F532 Mean
Mean feature pixel intensity at wavelength 532 nm.
F532 SD
The standard deviation of the feature pixel intensity at wavelength 532 nm.
B532 Median
The median background intensity at the wavelength 532 nm.
B532 Mean
The mean background intensity at the wavelength 532 nm.
B532 SD
The standard deviation of the local background intensity at wavelength 532 nm.
% > B532+1SD
The percentage of feature pixels with intensities more than one standard deviation above the background intensity at the wavelength 532 nm.
% > B532+2SD
The percentage of feature pixels with intensities more than two standard deviation above the background intensity at the wavelength 532 nm.
F532 % Sat.
The percentage of the feature pixels at wavelength 532 nm that are saturated.
Ratio of Medians (635/532)
The ratio of median intensities of each feature for wavelengths 635 nm and 532 nm, with the median background intensity subtracted.
Ratio of Means (635/532)
The ratio of the arithmetic mean intensities of each feature for wavelengths 635 nm and 532 nm, with the median background intensity subtracted.
Median of Ratios (635/532)
The median of pixel-by-pixel ratios of pixel intensities for wavelengths 635 nm and 532 nm, with the median background intensity subtracted.
Mean of Ratios (635/532)
The geometric mean of the pixel-by-pixel ratios of pixel intensities for wavelengths 635 nm and 532 nm, with the median background intensity subtracted.
Ratios SD (635/532)
The standard deviation of the log of pixel intensity ratios for wavelengths 635 nm and 532 nm.
Rgn Ratio (635/532)
The regression ratio for wavelengths 635 nm and 532 nm.
Rgn R² (635/532)
The coefficient of determination for the current regression value for the wavelengths 635 nm and 532 nm.
F Pixels
The total number of feature pixels.
B Pixels
The total number of local background pixels.
Sum of Medians
The sum of the median intensities for each wavelength, with the median background intensity at each wavelength subtracted.
Sum of Means
The sum of the arithmetic mean intensities for each wavelength, with the median background intensity at each wavelength subtracted.
Log Ratio (635/532)
log2 transform of the ratio of the medians for wavelengths 635 nm and 532 nm.
F635 Median - B635
The median feature pixel intensity at wavelengt 635 nm with the median background intensity subtracted.
F532 Median - B532
The median feature pixel intensity at wavelengt 532 nm with the median background intensity subtracted.
F635 Mean - B635
The mean feature pixel intensity at wavelengt 635 nm with the median background intensity subtracted.
F532 Mean - B532
The mean feature pixel intensity at wavelengt 532 nm with the median background intensity subtracted.
F635 Total Intensity
The sum of the feature pixel intensities at wavelength 635 nm.
F532 Total Intensity
The sum of the feature pixel intensities at wavelength 532 nm.
SNR 635
The signal-to noise ratio at wavelength 635 nm.
SNR 532
The signal-to noise ratio at wavelength 532 nm.
Flags
Flag used to mark the quality of the feature.
Normalize
Flag used to mark features to be used for normalization.
