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Journal of immunological methods2007; 328(1-2); 59-69; doi: 10.1016/j.jim.2007.08.006

Validation of quantitative polymerase chain reaction assays for measuring cytokine expression in equine macrophages.

Abstract: The study of the equine immune system and inflammatory responses, by measuring cytokine expression, can provide important insight into disease pathogenesis in the horse. A set of quantitative real-time polymerase chain reaction (QPCR) assays for the equine cytokines IL-1alpha, IL-1beta, IL-6, IL-8 and TNF-alpha were validated using QPCR primers and probes which were generated for the equine IL-1alpha, IL-1beta, IL-6, IL-8, TNF-alpha and 18S genes. Amplification efficiency, intra-assay and inter-assay variation were determined using 10-fold dilutions of plasmid for each gene. Under these conditions the amplification efficiencies of the primers and probes ranged from 99% to 101%. The mean coefficient of variation (CV) across five sets of plasmid DNA for both intra-assay and inter-assay variation was 0.63% (range 0.2% to 1.8%). Amplification efficiency was also determined using 2-fold dilutions of cDNA and under these conditions amplification efficiency ranged from 83% to 95%. The specificity of amplification was confirmed by DNA sequencing of reaction products. The QPCR assays were also evaluated using three sets of cDNA from equine monocyte derived macrophages (EMDM) stimulated for 1 h with lipopolysaccharide (LPS). The general trend was the same for all three samples with IL-1alpha showing the greatest induction and IL-6 the lowest induction. The range of cytokine induction was greater than has previously been reported with values ranging from 12-fold to 30,000-fold. We present a set of QPCR primers and probes that are suitable for quantitation of expression of a set of equine cytokines. The primers and probes have been rigorously analyzed, and we demonstrate that they are specific for the desired genes, have a high amplification efficiency and the assays are highly reproducible.
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Publication Date: 2007-09-04 PubMed ID: 17900609PubMed Central: PMC2148255DOI: 10.1016/j.jim.2007.08.006Google Scholar: Lookup
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  • Journal Article
  • Research Support
  • N.I.H.
  • Extramural
  • Research Support
  • U.S. Gov't
  • Non-P.H.S.
  • Validation Study

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research focused on validating a set of quantitative real-time polymerase chain reaction (QPCR) tests for measuring equine cytokine expression, which is a crucial part of studying horse immune system and inflammatory responses.

Explanation of the Research

The research focused on developing and validating a set of quantitative real-time polymerase chain reaction (QPCR) tests that can measure the expression of cytokines in the immune cells of horses. This can generate crucial insights into the pathogenesis of various diseases in horses.

  • The tests were validated using QPCR primers and probes synthesized for the cytokine genes IL-1alpha, IL-1beta, IL-6, IL-8, and TNF-alpha, along with the 18S gene.
  • To determine the amplification efficiency and variation within and between the tests, the researchers used 10-fold dilutions of plasmid corresponding to each gene. The amplification efficiencies for the probes and primers used ranged from 99% to 101%, indicating high efficiency.
  • The mean coefficient of variation (CV) across the trials for both within and between variations was 0.63%, indicating high reliability of the tests.

Evidence of Specificity and Efficiency

The researchers presented evidence of the tests’ efficiency and specificity.

  • Amplification efficiency was also determined using 2-fold dilutions of cDNA with results that ranged from 83% to 95%.
  • To confirm the specificity of amplification, the researchers sequenced the DNA products of the reactions. This allowed them to verify that the reactions were targeting the correct genes.

Practical Application

The QPCR tests were also applied in practice on cDNA from equine monocyte derived macrophages (EMDM) stimulated with lipopolysaccharide (LPS).

  • Three sets of EMDM were examined, with IL-1alpha showing the greatest amplification and IL-6 showing the least. The range of cytokine induction was greater than previously reported, ranging from 12-fold to 30,000-fold.
  • This demonstrated that the developed QPCR tests were not only efficient and specific, but also applicable in practice for measuring cytokine expression in equine immune cells.

Conclusion

In conclusion, a new set of QPCR primers and probes suitable for quantifying the expression of equine cytokines was presented. The set had been rigorously tested, and the research demonstrated that it specifically targets the intended genes, presents high amplification efficiency, and is highly reproducible.

Cite This Article

APA
Allen CA, Payne SL, Harville M, Cohen N, Russell KE. (2007). Validation of quantitative polymerase chain reaction assays for measuring cytokine expression in equine macrophages. J Immunol Methods, 328(1-2), 59-69. https://doi.org/10.1016/j.jim.2007.08.006

Publication

Journal of immunological methods
ISSN: 0022-1759
NlmUniqueID: 1305440
Country: Netherlands
Language: English
Volume: 328
Issue: 1-2
Pages: 59-69

Researcher Affiliations

Allen, Charlotte A
  • Department of Veterinary Pathobiology, Texas A&M University, MS 4467, College Station, TX 77843-4467, USA.
Payne, Susan L
    Harville, Melissa
      Cohen, Noah
        Russell, Karen E

          MeSH Terms

          • Animals
          • Cytokines / analysis
          • Cytokines / biosynthesis
          • DNA Primers / genetics
          • Gene Expression
          • Gene Expression Profiling / methods
          • Gene Expression Profiling / veterinary
          • Horses
          • Macrophages / immunology
          • RNA, Messenger / analysis
          • Reproducibility of Results
          • Reverse Transcriptase Polymerase Chain Reaction / methods
          • Reverse Transcriptase Polymerase Chain Reaction / veterinary

          Grant Funding

          • R01 CA059278 / NCI NIH HHS
          • R01 CA059278-11 / NCI NIH HHS
          • CA059278 / NCI NIH HHS

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          Citations

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