Application of QuantiGene nucleic acid quantification technology for high throughput screening.
To identify inhibitors of interleukin-8 (IL-8) production, a high throughput assay was developed using the QuantiGene nucleic acid quantification kit that employs branched-chain DNA (bDNA) technology to measure the mRNA directly from cells. Unlike polymerase chain reaction and other technologies that employ target amplification, the QuantiGene system uses signal amplification. To perform the assay, various molecular probes capable of hybridizing with IL-8 mRNA were designed and synthesized. A human lung epithelial cell line was treated with interleukin-1alpha (IL-1alpha) to stimulate the IL-8 gene expression and the mRNA was measured using the QuantiGene system. The QuantiGene assay was sensitive, flexible, and reproducible and achieved equivalent or better sensitivity than promoter-reporter assays, and eliminated the time required for constructing a promoter-reporter system. Our data show that bDNA technology has the potential to be used as a high throughput screening assay.
Warrior
U
U
Abbott Laboratories, Abbott Park, IL 60064, USA. usha.warrior@abbott.com
Fan
Y
Y
David
C A
CA
Wilkins
J A
JA
McKeegan
E M
EM
Kofron
J L
JL
Burns
D J
DJ
D021121:Branched DNA Signal Amplification Assay / Q000379:methods*; D002452:Cell Count; D002460:Cell Line; D015342:DNA Probes / Q000235:genetics; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D007118:Immunoassay; D007375:Interleukin-1 / Q000494:pharmacology; D016209:Interleukin-8 / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D008163:Luminescent Measurements; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors
D015342:DNA Probes; D007375:Interleukin-1; D016209:Interleukin-8; D012333:RNA, Messenger; D011933:Reagent Kits, Diagnostic