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

Hoadley, Margaret E.; Hopkins, Stephen J.

doi:10.1016/j.jim.2003.08.009

Cited by 4 publications

(2 citation statements)

References 26 publications

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“…This inclusion of most of the potential sources of variability in our analysis likely increased the reported RPD values. Nonetheless, the exhibited RPD values are well within the range of other interassay reproducibility values, ranging from 1 to 24% coefficient of variance (CV), reported in gene expression studies (Reddy and Wilkie, ; Hoadley and Hopkins, ; Cui et al, ; Allen et al, ; Pathak et al, ). Even the intra‐assay reproducibility values reported in literature (typically replicate wells or arrays with the same starting material), can be in similar range of up to 17% CV (Hoadley and Hopkins, ; Cui et al, ; Allen et al, ; Pathak et al, ).…”

Section: Discussionsupporting

confidence: 80%

An In Vitro alveolar macrophage assay for the assessment of inflammatory cytokine expression induced by atmospheric particulate matter

Sijan

Antkiewicz

Heo

et al. 2014

Environmental Toxicology

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Exposures to air pollution in the form of particulate matter (PM) can result in excess production of reactive oxygen species (ROS) in the respiratory system, potentially causing both localized cellular injury and triggering a systemic inflammatory response. PM-induced inflammation in the lung is modulated in large part by alveolar macrophages and their biochemical signaling, including production of inflammatory cytokines, the primary mechanism via which inflammation is initiated and sustained. We developed a robust, relevant, and flexible method employing a rat alveolar macrophage cell line (NR8383) which can be applied to routine samples of PM from air quality monitoring sites to gain insight into the drivers of PM toxicity that lead to oxidative stress and inflammation. Method performance was characterized using extracts of ambient and vehicular engine exhaust PM samples. Our results indicate that the reproducibility and the sensitivity of the method are satisfactory and comparisons between PM samples can be made with good precision. The average relative percent difference for all genes detected during 10 different exposures was 17.1%. Our analysis demonstrated that 71% of genes had an average signal to noise ratio (SNR) ≥ 3. Our time course study suggests that 4 h may be an optimal in vitro exposure time for observing short-term effects of PM and capturing the initial steps of inflammatory signaling. The 4 h exposure resulted in the detection of 57 genes (out of 84 total), of which 86% had altered expression. Similarities and conserved gene signaling regulation among the PM samples were demonstrated through hierarchical clustering and other analyses. Overlying the core congruent patterns were differentially regulated genes that resulted in distinct sample-specific gene expression "fingerprints." Consistent upregulation of Il1f5 and downregulation of Ccr7 was observed across all samples, while TNFα was upregulated in half of the samples and downregulated in the other half. Overall, this PM-induced cytokine expression assay could be effectively integrated into health studies and air quality monitoring programs to better understand relationships between specific PM components, oxidative stress activity and inflammatory signaling potential.

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Section: Discussionsupporting

confidence: 80%

An In Vitro alveolar macrophage assay for the assessment of inflammatory cytokine expression induced by atmospheric particulate matter

Sijan

Antkiewicz

Heo

et al. 2014

Environmental Toxicology

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“…All RNA samples for IL-1β mRNA measurements were routinely treated with 0.5 units RQ1 DNase (Promega, UK) per 50 ng total RNA at 37°C for 30 mins. The IL-1β, IL-6 and TNF-α mRNA in these samples were measured using an immunometric, reverse transcriptase, polymerase chain reaction, as previously described [22]. Where IL-6 and TNF-α mRNA values were high, these were also DNase treated to, although in no case were results substantially reduced by this treatment.…”

Section: Methodsmentioning

confidence: 99%

Clinical outcome following acute ischaemic stroke relates to both activation and autoregulatory inhibition of cytokine production

et al. 2007

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Background: As critical mediators of local and systemic inflammatory responses, cytokines are produced in the brain following ischaemic stroke. Some have been detected in the circulation of stroke patients, but their role and source is unclear. Focusing primarily on interleukin(IL)-1-related mechanisms, we serially measured plasma inflammatory markers, and the production of cytokines by whole blood, from 36 patients recruited within 12 h and followed up to 1 year after acute ischaemic stroke (AIS).

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Comparison of ‘real-time’ and immunometric RT-PCR: RNA interference of reverse transcriptase-PCR

Hoadley

Hopkins

2006

Journal of Immunological Methods

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

Cited by 4 publications

References 26 publications

An In Vitro alveolar macrophage assay for the assessment of inflammatory cytokine expression induced by atmospheric particulate matter

An In Vitro alveolar macrophage assay for the assessment of inflammatory cytokine expression induced by atmospheric particulate matter

Clinical outcome following acute ischaemic stroke relates to both activation and autoregulatory inhibition of cytokine production

Comparison of ‘real-time’ and immunometric RT-PCR: RNA interference of reverse transcriptase-PCR

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