Use of bidirectional blots in differential display analysis.
We have used bidirectional transfer methods in concert with SMART total cDNA complex probes to sequentially screen differential display arrays. In this report we show the utility of this methodology in examining a manganese superoxide dismutase cDNA fragment which we detected while evaluating the effects of the proinflammatory cytokines IL1-beta, TNF-alpha, and IL6 on human umbilical vein endothelial cell (HUVEC) gene expression. By using parallel hybridization of the bidirectional blots with SMART total cDNA (32)P probes derived from untreated or cytokine-treated HUVECs, differential expression between cell treatments can be clearly evaluated. Subsequent screening using this bidirectional blot method results in detection of modulated cDNA clones. Northern and total cDNA blot hybridization with the cDNA clonal fragment confirmed both modulated expression and the efficacy of this screening method. These procedures allow one to use bidirectional blots to evaluate band modulation on agarose gels which are initially run to evaluate the reamplification of display fragments or to confirm cloned cDNA fragments. Thus, bidirectional blot analysis using SMART total cDNA probes allows direct evaluation of differential display bands from the initial reamplification through plasmid insert cloning, increasing the investigator's ability to eliminate false-positive bands during each step of analysis.
Kestler
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Graduate School of Medicine, University of Tennessee Medical Center at Knoxville, Knoxville, Tennessee, 37920, USA. dkestler@utk.edu
Hill
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Agarwal
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Hall
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D015152:Blotting, Northern; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D018076:DNA, Complementary / Q000032:analysis; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004730:Endothelium, Vascular / Q000201:enzymology; D006801:Humans; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D013482:Superoxide Dismutase / Q000032:analysis / Q000235:genetics
D016207:Cytokines; D018076:DNA, Complementary; D013482:Superoxide Dismutase