genotype: mwh red ssdp[neo48] e l(3)?/+ tissue: Whole third instar male larvae
Growth protocol
Larvae were grown in small vials containing colored medium: 7.5g/l agar, 35g/l flour, 50g/l yeast, 55g/l glucose, 2.5ml/l p-Hydroxybenzoic Acid, 4ml/l Propionic Acid and 0.5ml/l Bromophenol. The colored medium allows for more precise staging of the larvae. Towards the end of the third larval stage the larvae cease to feed and the gut clears out. The colored medium can be seen through the live whole larvae. Larvae were collected when the gut was two thirds full and selected or the desired genotype using the GFP marker.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using Trizol (Life Technologies, Carlsbad, USA), followed by mRNA isolation using an Oligotex poly(A) extraction kit (Qiagen, Valencia, USA).
Label
Cy3
Label protocol
Purified mRNA (600 ng) was converted to either Cy3- or Cy5-labeled cDNA probes. Each reaction contains 50 mM Tris-HCl pH 8.3, 75 mM KCl, 15 mM MgCl2, 4 mM DTT, 2 mM dNTPs (0.5 mM each), 6 μg Cy3 or Cy5 random 9-mer (Trilink, San Diego, CA), 60 U RNase inhibitor (Ambion, Austin, TX), 600 U MMLV RT RNase (H-) (Promega, Madison, WI) in 75 μl. Labeled Cy3 or Cy5 cDNA products were combined and subsequently de-pooled into three aliquots and purified with ChromaSpin+ TE-30 gel-filtration spin column (Clontech). The probes were then re-pooled, concentrated by ethanol precipitation and resuspended in hybridization buffer.
genotype: w[*]/+; ssdp[BG1663]/mwh red ssdp[neo48] e l(3)? tissue: Whole third instar male larvae
Growth protocol
Larvae were grown in small vials containing colored medium: 7.5g/l agar, 35g/l flour, 50g/l yeast, 55g/l glucose, 2.5ml/l p-Hydroxybenzoic Acid, 4ml/l Propionic Acid and 0.5ml/l Bromophenol. The colored medium allows for more precise staging of the larvae. Towards the end of the third larval stage the larvae cease to feed and the gut clears out. The colored medium can be seen through the live whole larvae. Larvae were collected when the gut was two thirds full and selected or the desired genotype using the GFP marker.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using Trizol (Life Technologies, Carlsbad, USA), followed by mRNA isolation using an Oligotex poly(A) extraction kit (Qiagen, Valencia, USA).
Label
Cy5
Label protocol
Purified mRNA (600 ng) was converted to either Cy3- or Cy5-labeled cDNA probes. Each reaction contains 50 mM Tris-HCl pH 8.3, 75 mM KCl, 15 mM MgCl2, 4 mM DTT, 2 mM dNTPs (0.5 mM each), 6 μg Cy3 or Cy5 random 9-mer (Trilink, San Diego, CA), 60 U RNase inhibitor (Ambion, Austin, TX), 600 U MMLV RT RNase (H-) (Promega, Madison, WI) in 75 μl. Labeled Cy3 or Cy5 cDNA products were combined and subsequently de-pooled into three aliquots and purified with ChromaSpin+ TE-30 gel-filtration spin column (Clontech). The probes were then re-pooled, concentrated by ethanol precipitation and resuspended in hybridization buffer.
Hybridization protocol
Hybridization was performed in custom-made chambers allowing simultaneous exposure of the probe solution to both slides representing the entire Drosophila transcriptome. Spots (1 μl each) of a 40% suspension (v/v) of 30 μm ceramic microspheres in water were placed at four locations along each side of one slide and allowed to dry. The second slide was placed over the first slide such that the spotted parts of the slides were facing each other and the beads maintained proper spacing between the slides. Hybridization solution was applied at one end and covered the array surfaces by capillary action. Hybridization of labeled cDNA probes was performed in 50 μl 5x SSC, 0.1% SDS, and 1 mM DTT at 60°C for 6 h. The microarrays were washed after hybridization in 1x SSC, 0.1% SDS, 1 mM DTT at 45°C for 10 min, and then in 0.1x SSC, 0.2% SDS, 1 mM DTT at room temperature for 3 min. Microarrays were dried by centrifugation.
Scan protocol
Arrays were scanned using an Axon GenePix 3000A fluorescence reader (Molecular Devices Corporation, Union City, USA). GenePix v.4.1 image acquisition software (Molecular Devices Corporation) was used to extract signal for each target element.
Description
In the matrix file the data from slide A and B are combined, A is on top.
Data processing
The array data was analyzed using R. The raw intensity data normalized within-arrays using the PrintTipLoess algorithm, and next between-arrays, using Quantile. Array elements whose intensity was lower than the median intensity in both channels were discarded. No other background correction method was used. Duplicated elements were not averaged.
