For overexpression of otd, the hsp-otd line 5A generated by Royet and Finkelstein was used [Royet et al.: Pattern formation in Drosophila head development: the role of the orthodenticle homeobox gene. Development 1995; 121:3561-3572.]. All fly stocks were kept on standard cornmeal/yeast/agar medium at 25°C. Embryos were collected overnight for 12 hours on grape juice plates, further kept for 4 hours at 25°C and then subjected to a 37°C heat shock for 25 min, followed by a recovery period of 25 min at 25°C before RNA isolation. Therefore, at the time of RNA isolation these embryos were at embryonic stages 10-17 [Leemans et al.: Quantitative transcript imaging in normal and heat-shocked Drosophila embryos by using high-density oligonucleotide arrays. Proc Natl Acad Sci U S A 2000; 97:12138-12143.]. Embryos younger than embryonic stage 10 were not used, since heat shock in these earlier stages results in lethality [Walter set al.: Heat shock causes the collapse of the intermediate filament cytoskeleton in Drosophila embryos. Dev Genet 1990; 11:270-279.]
Total RNA was isolated from 200 mg of embryonic tissue, using guanidinium isothiocyanate in combination with acidic phenol (pH 4.0) (fast RNA tube green kit from BIO101) in a fast prep homogenizer FP120 (BIO 101). After precipitation, the RNA was dissolved in DEPC-treated water (Ambion) and spectrophotometrically quantified using a GeneQuant RNA/DNA calculator (Pharmacia Biotech). cDNA was synthesized upon total RNA as a template, using the SuperScript Choice System for cDNA synthesis (Gibco/BRL) with a T7-(T)24 DNA primer. This primer (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24VN-3') was purified by PAGE. For first-strand cDNA synthesis, a typical 40 ul reaction contained 25 ug RNA, 200 pmol T7-(T)24 primer, 500 uM of each dNTP and 800 units reverse transcriptase (AMV Superscript II). The reaction was incubated for 1 h at 42°C. Second-strand cDNA synthesis was carried out at 18°C for 2 h in a total volume of 340 ul, using 20 units Escherichia coli DNA ligase, 80 units E. coli DNA polymerase I and 4 units RNase H in the presence of 250 uM of each dNTP. After second-strand cDNA synthesis, 0.5 ul RNase A (100 mg/ml) (Qiagen) was added and the samples were incubated at 37°C for 30 min. Thereafter, 7.5 ul proteinase K (10 mg/ml) (Sigma) was added and the samples were further incubated at 37°C for another 30 min. After cDNA synthesis was completed, samples were phenol-chloroform extracted, using Phase Lock Gel (5 Prime-3 Prime) and ethanol precipitated. Biotinylated antisense cRNA was synthesized from the dsDNA template, using T7 RNA polymerase (MEGAscript T7 Kit: Ambion.). A 20 ul reaction volume contained between 0.3-1.5 ug cDNA, 7.5 mM of both ATP and GTP, 5.6 mM of both UTP and CTP and 1.8 mM of both biotinylated Bio-16-UTP and Bio-11-CTP (ENZO diagnostics) and 2 ul 10x T7 enzyme mix. The reaction was incubated at 37°C for 8 h. Thereafter, the unincorporated NTPs were removed by running the sample over an RNeasy spin column (Qiagen). Samples were precipitated, taken up in 20 ul DEPC-treated water and spectrophotometrically quantified. Thereafter, 40 ug of the biotinylated antisense cRNA was fragmented by heating the sample to 95°C for 35 min in a volume of 25 ul, containing 40 mM tris-acetate (pH 8.1), 100 mM potassium acetate, 30 mM magnesium acetate. After the fragmentation, the samples were placed on ice. Gene Chips were pre-hybridized with 220 ul hybridization buffer (1x MES (pH 6.7), 1 M NaCl, 0.01% Triton, 0.5 g/l acetylated BSA, 0.5 g/l sonicated herring sperm DNA) for 15 min at 45°C on a rotisserie (Heidolph) at 60 rpm. Hybridization was done in a final volume of 220 ul hybridization buffer, containing 40 g fragmented biotinylated cRNA. The samples were heated to 95°C for 5 min and briefly spun down. Hybridizations were carried out for 16 h at 45°C with mixing on a rotisserie at 60 rpm. After hybridization, the arrays were briefly rinsed with 6x SSPE-T (0.9 M NaCl, 0.06 M NaH2PO4, 6 mM EDTA, 0.01% Triton) and washed on a Fluidics station (Affymetrix). Hybridized arrays were stained with 220 ul detection solution (1x MES buffer, containing 2.5 ul streptavidin-R phycoerythrin conjugate (1 mg/ml) (Molecular Probes)) and 2.0 mg/ml acetylated BSA (Sigma) at 40°C for 15 min and washed again before being scanned.
The data was normalized against the mean of the total sums of Avg Diff values across all 16 arrays used in this study.
