Mice were kept on an isogenic 129SvJ background. Embryos were obtained from timed pregnancies and dissected at E10.5. 4 dual-color DNA-chip hybridizations of cDNAs from RNA pools of wt and homozygous Dll1tm1Gos embryos respectively were made. Total RNA from pooled samples was obtained according to manufacturer’s protocols using RNeasy Mini or Midi kits (Qiagen). 20µg total RNA was used for reverse transcription and indirectly labelled with Cy3 or Cy5 fluorescent dye according the TIGR protocol. Labelled cDNA was dissolved in 30µl hybridisation buffer (6xSSC, 0,5%SDS 5xDenhardt’s solution and 50% formamide) and mixed with 30µl of reference cDNA solution labelled with the second dye. This hybridisation mixture was placed on a pre-hybridised microarray, covered with cover slip, placed into a hybridization chamber (Gene Machines) and immersed in a thermostatic bath at 42°C for 18-20 hours. Slides were scanned for both Cy3 and Cy5 with a GenePix 4000A and images were processed by GenePix Pro 3.0. Mean intensities with subtracted background were normalised so that the median of their ratios has become 1.
