Application of QuantiGene™ Nucleic Acid Quantification Technology for High Throughput Screening

Abstract

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.

Effect of bupropion on CYP2B6 and CYP3A4 catalytic activity, immunoreactive protein and mRNA levels in primary human hepatocytes: comparison with rifampicin

“…We compared mRNA levels of treated cells to levels in vehicle control cells using a novel method of analysing mRNA levels in primary human hepatocytes based on the QuantiGene kit from Bayer, which may be as sensitive as RT-PCR for mRNA quantification (Hartley & Klaassen 2000;Warrior et al 2000;Zhou et al 2000;Sen et al 2001). The kit has an ELISA-like format and provides a solid support phase which binds RNA via probes specific for the gene of interest (capture extenders).…”

Twenty-Five Years of Quantitative PCR for Gene Expression Analysis

“…This approach offers the promise of a high-throughput assay. The Quantigene technology (Panomics, Fremont, CA, USA) (46) is a non-PCRbased technology that has been commercialized by Panomics. This technology differs from qPCR in that there is no PCR amplification; rather, capture and amplifier probes are used in conjunction with a chemilumigenic substrate.…”

X-FISH: Analysis of cellular RNA expression patterns using flow cytometry

“…Currently, there are two main kits available and being published with, supplied by EMD Millipore (SmartFlare TM ; (Khare et al, 2014;Seftor et al, 2014;Vaidyanathan et al, 2014)) and eBioscience (PrimeFlow TM RNA and QuantiGene® FlowRNA (Warrior et al, 2000;Zhang et al, 2005;Canales et al, 2006)). The biggest advantage of the SmartFlare technology is that it can be utilized on live cells, allowing these to be studied over time and in various downstream live-cell assays.…”