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PubMed · 14604547

Simple, quantitative measurement of cytokine gene expression using an immunometric reverse transcriptase-polymerase chain reaction.

Margaret E Hoadley·Stephen J Hopkins
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Abstract

We have developed a novel system to quantify mRNA, using a coupled reverse transcriptase-polymerase chain reaction (RT-PCR) with RNA standards and an immunometric optical readout. Biotinylated RT-PCR products were captured onto avidin-coated microplates and hybridised to a dinitrophenol (DNP)-labelled oligonucleotide probe. Enzyme-linked anti-DNP antibodies were used to detect the product via a colorimetric enzyme assay. Unknown mRNA samples were quantified by interpolating the signal on a standard curve generated from dilutions of RNA (cRNA) standards for interleukin-6 (IL-6), tumour necrosis factor-alpha (TNFalpha), IL-1beta and actin mRNA. The assay for IL-6 mRNA was able to discriminate between samples with a two- to fourfold difference in concentration, had an intra-assay coefficient of variation (CV) of 17% and an inter-assay CV of 24%. The method was used to quantify IL-6 mRNA, relative to expression of IL-6 protein and biological activity, in human blood that had been activated by endotoxin. The IL-6 mRNA could be detected 1 h after endotoxin addition and peaked at 4 h. This paralleled the increase in protein production that followed approximately 1 h later.

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Cell LineCytokinesEnzyme-Linked Immunosorbent AssayHumansInterleukin-6LipopolysaccharidesRNA, MessengerReproducibility of ResultsReverse Transcriptase Polymerase Chain ReactionJournal Article

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Margaret E Hoadley, Stephen J Hopkins. 2003. Simple, quantitative measurement of cytokine gene expression using an immunometric reverse transcriptase-polymerase chain reaction.. https://doi.org/10.1016/j.jim.2003.08.009

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