strain/genotype: UAS-GFP:Su(H) expressed by the patched[559.1]-Gal4 driver developmental stage: third instar lavae tissue: Wing imaginal discs
Growth protocol
Larvae grown at 25C.
Extracted molecule
total RNA
Extraction protocol
Wing imaginal discs form 60 third instar larvea were dissected for each RNA isolation using Trizol (Sigma).
Label
Cy3
Label protocol
To 3 to 5 µg of total RNA, add 1 µl 500 ng/ µl of Oligo(dT)23 anchored primer (Sigma) and adjust volume to 5 µl with DEPC- water. Incubate at 65 °C for 10 min, then snap cool on ice. Add 4 µl 5x First Strand Buffer (Invitrogen), 2 µl 0.1M DTT (Invitrogen), 1 µl 10 mM dNTP mix, 0.25 µl 40 U/µl RNasin (Promega) and 0.75 µl 200 U/µl Superscript III (Invitrogen). Incubate at 46 °C for 2 hours, then at 65 °C for 15 minutes, snap cool on ice. For second strand synthesis: Add 7.5 µl Second Strand Buffer (Invitrogen), 0.75 µl 10 mM dNTP mix, 2 µl 10 U/µl DNA Polymerase I (Invitrogen), 0.1 µl 5 U/µl RnaseH (Promega), 0.5 µl 10 U/µl E. coli Ligase (NEB) adjust volume to 40 µl with DEPC-water. Incubate at 16 °C for 2 hours, then purification using QIAquick PCR Purification Kit. To label take up to 1 µg double stranded DNA and make up to a total volume of 25 µl with DEPC-water. Add 20 µl 2.5x Random Primer Reaction Buffer (Invitrogen – BioPrime DNA Labelling Kit). Incubate at 100 °C for 5 minutes, snap cool on ice. Add 1 µl 10 X low-C dNTP mix (5mM dATP,dGTP,dTTP, 2 mM dCTP), 2 µl 1 mM Cy3 or Cy5 dCTP (GE Healthcare) and 1 µl 40U/µl Klenow (Invitrogen – BioPrime DNA Labelling Kit). Incubate at 37°C for 2 to 3 hours. Stop the reaction by adding 5 µl Stop Buffer (Invitrogen – BioPrime DNA Labelling Kit). Resuspend the resin in the AutoSeq G-50 column (GE Healthcare) by vortexing gently. Loosen the cap a quarter turn and snap off the bottom closure. Place the column in a collection tube. Pre-spin column at 5,000 rpm for 1 minute to remove the buffer. Remove the top cap and place column in a new 1.5 ml tube. Pipette half of the sample onto the centre of the angled surface of the compacted resin bed being careful not to disturb the resin. Spin for 1 minute at 5,000 rpm. Discard the column. Place a second column into the same 1.5 ml microfuge tube and then add the second half of the sample. Spin for 1 minute and 5,000 rmp. Reduce volume of probe to between 2 to 5 µl by placing in a speed vac with medium heat. Add 2 µl of 10 mg / ml sonicated salmon sperm DNA (Invitrogen).
strain: UAS-NLS-GFP expressed by the patched[559.1]-Gal4 driver developmental stage: third instar lavae tissue: Wing imaginal discs
Growth protocol
Larvae grown at 25C.
Extracted molecule
total RNA
Extraction protocol
Wing imaginal discs form 60 third instar larvea were dissected for each RNA isolation using Trizol (Sigma).
Label
Cy5
Label protocol
To 3 to 5 µg of total RNA, add 1 µl 500 ng/ µl of Oligo(dT)23 anchored primer (Sigma) and adjust volume to 5 µl with DEPC- water. Incubate at 65 °C for 10 min, then snap cool on ice. Add 4 µl 5x First Strand Buffer (Invitrogen), 2 µl 0.1M DTT (Invitrogen), 1 µl 10 mM dNTP mix, 0.25 µl 40 U/µl RNasin (Promega) and 0.75 µl 200 U/µl Superscript III (Invitrogen). Incubate at 46 °C for 2 hours, then at 65 °C for 15 minutes, snap cool on ice. For second strand synthesis: Add 7.5 µl Second Strand Buffer (Invitrogen), 0.75 µl 10 mM dNTP mix, 2 µl 10 U/µl DNA Polymerase I (Invitrogen), 0.1 µl 5 U/µl RnaseH (Promega), 0.5 µl 10 U/µl E. coli Ligase (NEB) adjust volume to 40 µl with DEPC-water. Incubate at 16 °C for 2 hours, then purification using QIAquick PCR Purification Kit. To label take up to 1 µg double stranded DNA and make up to a total volume of 25 µl with DEPC-water. Add 20 µl 2.5x Random Primer Reaction Buffer (Invitrogen – BioPrime DNA Labelling Kit). Incubate at 100 °C for 5 minutes, snap cool on ice. Add 1 µl 10 X low-C dNTP mix (5mM dATP,dGTP,dTTP, 2 mM dCTP), 2 µl 1 mM Cy3 or Cy5 dCTP (GE Healthcare) and 1 µl 40U/µl Klenow (Invitrogen – BioPrime DNA Labelling Kit). Incubate at 37°C for 2 to 3 hours. Stop the reaction by adding 5 µl Stop Buffer (Invitrogen – BioPrime DNA Labelling Kit). Resuspend the resin in the AutoSeq G-50 column (GE Healthcare) by vortexing gently. Loosen the cap a quarter turn and snap off the bottom closure. Place the column in a collection tube. Pre-spin column at 5,000 rpm for 1 minute to remove the buffer. Remove the top cap and place column in a new 1.5 ml tube. Pipette half of the sample onto the centre of the angled surface of the compacted resin bed being careful not to disturb the resin. Spin for 1 minute at 5,000 rpm. Discard the column. Place a second column into the same 1.5 ml microfuge tube and then add the second half of the sample. Spin for 1 minute and 5,000 rmp. Reduce volume of probe to between 2 to 5 µl by placing in a speed vac with medium heat. Add 2 µl of 10 mg / ml sonicated salmon sperm DNA (Invitrogen).
Hybridization protocol
Add 140 µl of Ocimum hybridisation buffer to the labelled mixture and heat at 100 °C for 2 minutes on a hot-block. Centrifuge for 3 minutes at 13,000 rpm. Load 140 µl of the labelled sample to the microarray, and hybridise for 16 hours at 51 °C. Perform post-hybridisation washes as per PowerMatrix slides protocol (FMB). Pre-heat wash solution 1 (0.2 x SSC; 0.2% SDS) and wash solution 2 (0.2 x SSC) to 55 °C using a water bath. Incubate slides in a slide staining dish with pre-heated wash solution 1 on an orbital shaker at 50 rpm for 20 minutes. Transfer the slides to a staining trough containing pre-heated wash solution 2. Gently dip the slides up and down for 1 minute in wash solution 2. Repeat step in fresh wash solution 2 twice. Rinse the slides 3 times with fresh deionized water at room temperature (dip the rack in MilliQ water for 3 seconds). Transfer the slides to a clean microscope slide box with tissue at the base and centrifuge at 1000 rpm for 5 minutes. The slides are now ready to be scanned.
Scan protocol
Arrays are scanned at 5 µm resolution with a GenePix 4000B (Axon) dual laser scanner at 100% Power, with Lines to average = 1 and Focus position = 0 at their respective optimal PMT gains selected by the Auto-PMT function (0.005% saturation tolerance). Images are saved as Single-image TIFF.
Description
biological replicate 3, dye-swap S109516
Data processing
Images are processed and spot quantified by the Dapple software (http://www.cs.wustl.edu/~jbuhler/research/dapple/). The R package limma (SMYTH and SPEED 2003) was used to normalize the arrays. For each array, the background intensity level of the array image was first adjusted before loess normalization was applied to the MA values of the array. The intensities of the two channels of all 3 arrays were then quantile normalized, such that the intensities all had the same empirical distribution. Missing values (i.e. reduced data set) arise because genes are not expressed or there is poor quality on the array. The resulting ratios are log2 ratio of sample/control.
