Simple, quantitative measurement of cytokine gene expression using an immunometric reverse transcriptase-polymerase chain reaction.
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.
Hoadley
Margaret E
ME
University of Manchester Injury Research Group, Clinical Sciences Building, Hope Hospital, Salford M6 8HD, UK. mhoadley@fs 1.ho.man.ac.uk
Hopkins
Stephen J
SJ
D002460:Cell Line; D016207:Cytokines / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D016207:Cytokines; D015850:Interleukin-6; D008070:Lipopolysaccharides; D012333:RNA, Messenger