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.
D000180:Acrylic Resins / Q000737:chemistry; D000287:Administration, Topical; D000875:Anthralin / Q000494:pharmacology*; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D001483:Base Sequence; D018929:Cell Culture Techniques; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects* / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011565:Psoriasis / Q000235:genetics*; D018075:RNA, Complementary / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry / Q000145:classification; D016622:Silver Staining