Aida imaging software

For quantitative evaluation of gene expression levels, capillary Northern blot technique was applied according to the protocol for transfer onto nylon membranes at neutral pH, published in Sambrook and Russell (2001) . Subsequent labeling of specific RNA molecules was performed with digoxigenin-11-UTP (DIG-dUTP, Roche Diagnostics Deutschland GmbH, Mannheim, Germany)-labeled probes, generated by PCR (Supplementary Table S3). Labeled RNA was detected using anti-DIG antibodies and the substrate CDP-Star (both obtained from Roche Diagnostics Deutschland GmbH, Mannheim, Germany). Visualization and quantification was achieved by chemiluminescence using the imaging system Stella 3200 and AIDA imaging software (raytest, Straubenhardt, Germany).

cDNA microarrays of protease and protease inhibitor sequences on nylon membranes and the synthesis of digoxigenin labeled cDNA have been described previously [66 (link)]. Detailed information on the generation of the protease/protease inhibitor probes, their arrangement on the membranes as well as experimental details have been published [67 (link)]. In brief, cDNA prepared from COS-7 cells was digoxigenin-labeled and hybridized on a custom oligonucleotide microarray comprising housekeeping genes, positive and negative controls, and genes representing a collection of human intra- and extracellular proteases, and protease inhibitors. Hybridization patterns were subsequently detected by chemiluminescence and analyzed using the AIDA imaging software (Raytest, Straubenhardt, Germany). Average densitometry signals of duplicate spots from K-RasG12V/E1- and K-RasG12V/E1-R3-xpressing cells were corrected for the background and normalized against the respective signal from E1-expressing cells.