strain: Cousin Island (Jean R. David, Centre National de la Recherche Scientific, Gif sur Yvette, France) gender: Male stage: 96h post larval emergence
Treatment protocol
Flies were grown on standard cornmeal-molasses medium on 10 cm culture plates, painted with a small amount of water-diluted yeast. No more than 30 individuals were on a single plate to prevent growth-dependent density effects. When flies reached the appropriate stage, they were collected in RNAlater (Ambion) on ice and placed immediately at -80 degrees centigrade.
Extracted molecule
total RNA
Extraction protocol
mRNA was extracted from a pool of 25 males using the RNeasy Mini kit (Qiagen). Samples were then amplified twice using the MessageAmp II aRNA kit (Ambion).
Label
Alexa647
Label protocol
RNase-free water was added to 5 μg of total RNA from each sample to bring the final volume to 14.5 μl. 4 μl of random primer was added followed by incubation at 70°C for 10 min, then 42°C for 5 min. 19.5 μl of modified Indirect RT master mix was added to each tube, along with 2.0 μl of Superscript II RT and the reaction was incubated at 42°C for 3 hours. The cDNA product was cleaned and precipitated by adding 8 μl of 1N NaOH to each reaction with mixing by pipetting followed by a quick spin and immediate incubation at 65°C for 10 min. 8 μl of 1N HCl was then added, followed by 4 μL of 1M Tris (pH 7.5), mixing by pipetting after each addition. 38 μl of water was added to bring the total volume to 100 μl, and the amino allyl-cDNA was purified using either the Qiagen PCR clean up or Invitrogen Purelink purification kit (using 80% EtOH for the wash buffer and eluting with 2 × 50 μl of water). After purification, 10 μl of 3M NaOAc, 1 μl of glycogen (20 μg/μl) and 120 μl of ice-cold isopropanol were added, and the cDNA was allowed to precipitate at –20°C for at least 75 min or overnight. The precipitated cDNA was then spun at >12,000 g for 30 min and the pellet was washed with 200 μl of 75% EtOH, followed by another spin at >12,000 g for 5 min. All EtOH was carefully pipetted from the tube and the probe pellet was allowed to dry for ≤ 1 min before resuspension in 5 μl of water. Samples were then dye conjugated by addition of 3 μl of 0.3 M NaHCO3 to the resuspended amino allyl-cDNA, followed by 2 μl of reactive dye and subsequent incubation at room temperature in the dark for 1 hour. 90 μl of ddH2O was added to each sample, followed by purification using either the Qiagen PCR clean up or Invitrogen Purelink purification kit, washing with 80% EtOH 3 × and eluting with 3 × 50 μl of water. 15 μl of 3M NaOAc, 1.5 μl of glycogen (20 μg/μl) and 170 μl of ice-cold isopropanol were added to the labeled probe, and the DNA was allowed to precipitate at –20°C for at least 30 min. The precipitated probe was then spun at >12,000 g for 30 min and the pellet was washed with 200 μl of 75% EtOH, and spun at >12,000 g for 5 min. All EtOH was carefully pipetted from the tube and the probe pellet was allowed to dry for ≤ 1 min before resuspension in 5 μl of water.
Channel 2
Source name
D. melanogaster male mixed-stage whole-body mRNA reference
strain: 14021-0231.00 gender: Male stage: Equal concentration mix of 96h post larval emergence, 4h post puparium formation, 72h post puparium formation, and 1.5h post eclosion individuals.
Treatment protocol
Flies were grown on standard cornmeal-molasses medium on 10 cm culture plates, painted with a small amount of water-diluted yeast. No more than 30 individuals were on a single plate to prevent growth-dependent density effects. When flies reached the appropriate stage (synchronized to a 2h window), they were collected in RNAlater (Ambion) on ice and placed immediately at -80 degrees centigrade.
Extracted molecule
total RNA
Extraction protocol
mRNA was extracted from pools of 25 males at each stage listed above individually using the RNeasy Mini kit (Qiagen). A large number of extracts were performed for each stage. Within-stage extracts were pooled, quantified, and the four stages were mixed together in equal concentration in order to produce the reference sample.
Label
Alexa555
Label protocol
RNase-free water was added to 60 μg of total RNA from each sample, to bring the final volume to 19 μl. 21 μl of Indirect RT master mix was added into each tube and the reaction was incubated at 65°C for 5 min, then 42°C for 5 min. 2 μl of Superscript II RT was added to the sample, followed by incubation at 42°C for 3 hours. The cDNA product was cleaned and precipitated by adding 8 μl of 1N NaOH to each reaction with mixing by pipetting followed by a quick spin and immediate incubation at 65°C for 10 min. 8 μl of 1N HCl was then added, followed by 4 μL of 1M Tris (pH 7.5), mixing by pipetting after each addition. 38 μl of water was added to bring the total volume to 100 μl, and the amino allyl-cDNA was purified using either the Qiagen PCR clean up or Invitrogen Purelink purification kit (using 80% EtOH for the wash buffer and eluting with 2 × 50 μl of water). After purification, 10 μl of 3M NaOAc, 1 μl of glycogen (20 μg/μl) and 120 μl of ice-cold isopropanol were added, and the cDNA was allowed to precipitate at –20°C for at least 75 min or overnight. The precipitated cDNA was then spun at >12,000 g for 30 min and the pellet was washed with 200 μl of 75% EtOH, followed by another spin at >12,000 g for 5 min. All EtOH was carefully pipetted from the tube and the probe pellet was allowed to dry for ≤ 1 min before resuspension in 5 μl of water. Samples were then dye conjugated by addition of 3 μl of 0.3 M NaHCO3 to the resuspended amino allyl-cDNA, followed by 2 μl of reactive dye and subsequent incubation at room temperature in the dark for 1 hour. 90 μl of ddH2O was added to each sample, followed by purification using either the Qiagen PCR clean up or Invitrogen Purelink purification kit, washing with 80% EtOH 3 × and eluting with 3 × 50 μl of water. 15 μl of 3M NaOAc, 1.5 μl of glycogen (20 μg/μl) and 170 μl of ice-cold isopropanol were added to the labeled probe, and the DNA was allowed to precipitate at –20°C for at least 30 min. The precipitated probe was then spun at >12,000 g for 30 min and the pellet was washed with 200 μl of 75% EtOH, and spun at >12,000 g for 5 min. All EtOH was carefully pipetted from the tube and the probe pellet was allowed to dry for ≤ 1 min before resuspension in 5 μl of water.
Hybridization protocol
80 μL of hybridization buffer (75 μl of DIG Easy Hyb [Roche], 4 μl of 10 mg/ml yeast tRNA [Invitrogen], and 4 μl of 10 mg/ml salmon sperm DNA [Sigma]) was added to each resuspended probe, followed by incubation at 65°C for 10 min. Both sample and reference probes were placed on the array, which was then placed in a sealed chamber in a 37°C water bath for 16-18 hours. The array was then washed for 3 × 15 min in pre-warmed 1 × SSC, 0.1% SDS. The array was then washed with room temperature 1 × SSC for ≤1min, followed by room temperature 0.1 × SSC for ≤ 15 sec.
Scan protocol
Array was scanned using a ScanArray 4000 XL (GSI Lumonics/Packard Biochips); images were preprocessed and quantified using QuantArray v3.0 (PerkinElmer).
Description
All information is provided above.
Data processing
While the CH1_SIG_MEAN, CH1_BKD_MEAN, CH2_SIG_MEAN, and CH2_BKD_MEAN columns are derived from the raw data, the VALUE column was derived as follows:
Data from the scanned microarrays were uploaded into GeneTraffic™ DUO version 3.0 (Iobion Informatics) and replicate spots were filtered such that any element showing greater than 200% covariance or a 2-fold greater difference among the highest and lowest measured replicate spot (including all elements within an array or between replicate arrays) was flagged. All elements flagged according to these criteria as well as those flagged by the internal quality control standards of the software were then manually inspected for the presence of unacceptable spots (e.g., incorrectly printed, contained visible surface scratches or matter interfering with the scanning, etc.). Such spots were removed, and if less than 6 usable replicates (i.e., 2 replicate spots per array) remained, the entire element was discarded. Furthermore, an element was discarded if a subset of the within-array replicate spots showed consistently different hybridization intensities; these spots likely represent clones that were incorrectly annotated as belonging to the same element in the CDMC’s Drosophila 12kv2 microarray annotation file (http://142.150.8.217/GT_annot.zip). The filtered raw data was then downloaded from GeneTraffic™ DUO version 3.0 and subjected to a second round of quality control, where spots were removed if they did not show an expression intensity of at least 100, as well as a two-fold expression intensity above either the local or global average background. All genes that did not have usable data from both replicate spots on all three microarrays in all four stages within a species were then removed from further analysis. The CDMC’s Drosophila 12kv2 microarray annotation file was then manually inspected in order to identify all spots for which a single Flybase gene number (FBgn) (FB2008_10 Dmel Release 5.13; http://flybase.org/) could unambiguously be identified. All control spots as well ambiguous clone spots were removed from further analysis, leaving only genes that were detectably expressed in all 4 stages in at least one of the 3 species or the hybrid.
The data remaining after quality control were normalized using the ‘rlowess’ procedure (spatial-intensity joint loess) as implemented in the ‘maanova’ package in the R statistical software (http://research.jax.org/faculty/churchill/software/Rmaanova/index.html), using default values and a ‘TwoColor’ array type. The output for each spot on each array from maanova was then transformed into its log2(sample/reference) ratio, which is reported in the 'VALUE' column.
