Optimized visualization and PCR reamplification of differentially displayed cDNA bands detected by silver staining in polyacrylamide gels as established in the model of dithranol-treated keratinocytes.

The differential display of cDNA species defined by a combination of so-called anchored and arbitrary primers has been acknowledged as a powerful complex strategy to identify differences in gene expression, and depends in its original version, inaugurated by P. Liang and A. B. Pardee in 1992, on the use of radioactive-labelled nucleotides. As a non-radioactive methodological alternative, we established the use of polyesterfilm-backed 10% polyacrylamide gels for horizontal differential-display electrophoresis under non-denaturing conditions, with subsequent detection of cDNA bands by an optimized, semi-automated silver staining omitting any fixation step. Polyacrylamide gel slices carrying the silvered cDNA species of interest were cut out, chopped, squashed and incubated in an ammonium acetate/EDTA solution at 37 degrees C overnight under vigorous shaking. This procedure resulted in a 70% average success rate for subsequent PCR reamplification with regard to the number of cDNAs harvested from the differential-display gel. Novel sequence data of three cDNA clones are communicated, which under these methodological conditions were selected to be up- or down-regulated, respectively, by antipsoriatic dithranol in cultured HaCaT keratinocytes.