Experimental Design To identify gcm downstream genes in a comprehensive manner, we used genome-wide oligonucleotide arrays to analyse differential gene expression in Drosophila wild-type embryos versus embryos in which gcm is misexpressed throughout the neuroectoderm. The wild-type was Oregon-R. Tissue-specific misexpression was achieved by using virgin females from scabrous-GAL4 (Klaes et al., 1994) that were crossed to yw; UAS-gcm; UAS-gcm males (Jones et al., 1995). Transcripts were analysed at two defined temporal windows during embryogenesis. First, during a period of initial gcm action on determination of glial cell precursors and second during a later period when glial cells have already differentiated. After a 1 hr pre-collection, wild-type and sca-gcm embryos were collected in parallel for 1 hr and staged to 6-7 hrs AEL (stage 11) or to 13-14 hrs AEL (late stage 15/early stage 16). Stages are according to (Campos-Ortega and Hartenstein, 1997).
Samples used, extract preparation and labeling 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 (BIO101). 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 PAGE purified. For first-strand cDNA synthesis, a typical 20 μl reaction contained 20 μg total RNA, 100 pmol T7-(T)24 primer, 1 mM of each dNTP and 800 units reverse transcriptase (AMV Superscript II). The reaction was incubated for 1 hr at 42°C. Second-strand cDNA synthesis was carried out at 18°C for 2 hrs in a total volume of 150 μl, using 10 units E. coli DNA ligase, 40 units E. coli DNA polymerase I and 4 units Rnase A in the presence of 200 μM dNTPs. After second-strand cDNA synthesis, samples were incubated with 20 units T4 DNA polymerase at 18°C for 5 min. Then, 0.5 μl Rnase A 100 (μg/μl) (Quiagen) was added and the samples were incubated at 37°C for 30 min. Thereafter, 2.5 μl proteinase K (30 mg/ml) (Sigma) was added and the samples were further incubated at 37°C for another 30 min. After cDNA synthesis was completed, the samples were phenol-chloroform extracted using Phase Lock Gel (Eppendorf) and ethanol precipitated. Biotinylated antisense cRNA was synthesized from the dsDNA template, using T7 RNA polymerase (MEGAscript T7 Kit, Ambion). A 20 μl reaction volume contained between 0.3-1.5 μg 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 2μl 10x T7 enzyme mix. The reaction was incubated at 37°C for 6-8 hrs. Thereafter, the unincorporated dNTPs were removed by running the sample over an Rneasy spin column (Qiagen). Samples were precipitated, dissolved in 20 μl DEPC-treated water and spectrophotometrically quantified. Thereafter, 35 μg of the biotinylated antisense cRNA was fragmented by heating the sample to 95°C for 45 min in a volume of 25 μl, containing 40 mM tris-acetate (pH 8.1), 100 mM potassium acetate and 30 mM magnesium acetate. After fragmentation, the samples were placed on ice.
Hybridization procedures Gene Chips were prehybridized with 220 μl hybridization buffer ( 1x MES (pH 6.7), 1M NaCl, 0.01 % Tween 20, 0.5 μg/μl acetylated BSA, 0.5 μg/μl sonicated herring sperm DNA ) for 15 min at 40°C on a REAX 2 rotisserie (Heidolph, Swabach, Germany) at 60 rpm. Hybridization was done in a final volume of 220 μl hybridization buffer containing 35 μg fragmented biotinylated cRNA. The samples were heated to 95°C for 5 min and briefly spun down. Hybridization was carried out for 16 hrs at 45°C on a rotisserie at 60 rpm. After hybridization, the arrays were briefly rinsed with 6x SSPE-Tw (0.9 M NaCl, 0.06 M NaH2PO4, 6 mM EDTA, 0.01 % Tween 20) and washed on a Fluidics Station (Roche) for 5 min. Chips were rinsed with MES-wash buffer (1x MES, 26 mM NaCl, 0.01 % Tween) and then 230 μl fresh MES-wash-buffer was applied to wash the chips at 45°C for 30 min with rotation. Hybridized arrays were stained with 230 μl stain buffer (1xMES, 1M NaCl, with 2.3 μl streptavidin-R phycoerithrin conjugate (1 mg/ml) (Molecular Probes) and 2.5 μg/ml acetylated BSA (Sigma) at 40°C for 15 min and at 60 rpm. The chips were rinsed and washed again on a Fluidics Station (Roche) for 5 min before scanning.
Measurement data and data processing Probe arrays were scanned with a commercial confocal laser scanner (Hewlett-Packard). Pixel intensities were measured, and expression signals were analysed with commercial software (Genechip 4.0, Affymetrix).
