Margaret E Hoadley | PubMed Health

PubMed2006

Comparison of 'real-time' and immunometric RT-PCR: RNA interference of reverse transcriptase-PCR.

We measured interleukin-6 (IL-6) mRNA in whole blood, using an immunometric reverse transcriptase-polymerase chain reaction (IRT-PCR) method and 'real-time' RT-PCR using the Roche LightCycler. Both methods showed similar precision, in terms of inter- and intra-assay variation, where a lower total RNA concentration was used at a relatively high ratio of (IL-6) mRNA to total RNA. However, the 'real-time' RT-PCR method showed a greater loss of accuracy at higher concentrations of total RNA.

Humans↗

PubMed2003

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.

Cell Line↗