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Home / Equine Research Bank / J Immunol Methods / Quantitative measurement of equine cytokine mRNA expression ...
Journal of immunological methods1999; 222(1-2); 155-169; doi: 10.1016/s0022-1759(98)00193-8

Quantitative measurement of equine cytokine mRNA expression by polymerase chain reaction using target-specific standard curves.

Abstract: Quantification of cytokine mRNA using reverse transcription coupled with the polymerase chain reaction (RT-PCR) has become a corner stone of the study of cytokine regulation. Quantitative competitive RT-PCR (QCRT-PCR) is commonly accepted as a reliable method for quantifying differences in mRNA levels but is both labor- and reagent-intensive. A noncompetitive polymerase chain reaction method that utilizes cytokine-specific, plasmid-derived, standard curves was developed for the quantification of equine cytokine mRNA. The assay can be performed on minute samples of cellular material, utilizes sequences identical to wild-type for the generation of standard curves, is technically facile, less reagent-and labor-intensive than competitive methods, easily accommodates high sample throughput without the use of radioactive labels, and generates replicate samples to allow statistical analysis of the data. We demonstrate the utility of the assay, which is easily adapted to any cloned mRNA sequence, using equine interleukin-10 (IL-10). Both IL-10 and beta-actin cDNA were amplified in triplicate PCR reactions from oligo-dT primed RT reactions. Dilutions of plasmid DNA encoding the respective sequence, equine IL-10 or beta-actin, were also amplified in triplicate reactions in the same run. Beta-actin cycling parameters were modified to maintain the amplification in its exponential phase by decreasing both cycle number and cDNA volume relative to the parameters used for cytokine amplification. Following amplification, aliquots of the PCR reactions were hybridized with sequence-specific tris (2,2'-bipyridine) ruthenium II chelate labeled oligonucleotide probes and quantified using the QPCR System 5000. Plasmid derived values were used to generate a standard curve for the interpolation of mRNA content in unknown cDNA samples. Beta-actin values were used to derive a factor for the relative normalization of differences among cDNA samples that are inherent in the RNA extraction and RT steps. This assay resolves at least 2-fold differences in message, is reproducible, and has a dynamic range on the order of 3 logs.
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Publication Date: 1999-02-18 PubMed ID: 10022382DOI: 10.1016/s0022-1759(98)00193-8Google Scholar: Lookup
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  • Journal Article
  • Research Support
  • Non-U.S. Gov't
  • Research Support
  • U.S. Gov't
  • P.H.S.

Summary

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The study titled “Quantitative measurement of equine cytokine mRNA expression by polymerase chain reaction using target-specific standard curves” explores the development of a noncompetitive polymerase chain reaction technique. This method is utilized in quantifying the mRNA of equine cytokines, using cytokine-specific, plasmid-derived, standard curves and can be effectively performed on minute cellular sample material.

Objective of the Research

  • The research project aims to highlight an advanced methodology to quantify the mRNA levels of equine cytokines with a specific focus on its adaptation for equine interleukin-10 (IL-10).
  • What sets this polymerase chain reaction-based technique apart is its reduced dependency on intensive labour and reagents. Besides, it eliminates the need for any radioactive labels and facilitates high sample throughput.

Techniques and Methodology

  • The technique involves the use of reverse transcription along with the polymerase chain reaction (RT-PCR) to study cytokine regulation – specifically equine interleukin-10 (IL-10). With the use of these methods, researchers can quantitatively measure cytokine mRNA levels.
  • Standard curves, identical to wild-type sequences, were employed in the assay.
  • The generation of replicate samples facilitates an in-depth statistical analysis of the data.

Procedure and Analysis

  • Both IL-10 and beta-actin cDNA, essential for the procedure, were amplified in triplicate PCR reactions from oligo-dT primed RT reactions.
  • Following the amplification, sequence-specific probes were used to conduct a hybridization. It was followed by quantification using the QPCR System 5000.
  • The beta-actin values were used as a basis for deriving a factor essential for the relative normalization of differences among cDNA samples.

Findings of the Research

  • The discovery showcases the ability of the assay to resolve at minimum 2-fold differences in messages. It also demonstrates high reproducibility and a dynamic range on the order of 3 logs, showcasing this technique’s potential in cytokine mRNA measurement.
  • Thus, the research proves the utility of this noncompetitive PCR method in quantifying equine cytokine mRNA, offering a reliable, less-resource demanding, and easily adaptable option to study cytokine regulation.

Cite This Article

APA
Swiderski CE, Klei TR, Horohov DW. (1999). Quantitative measurement of equine cytokine mRNA expression by polymerase chain reaction using target-specific standard curves. J Immunol Methods, 222(1-2), 155-169. https://doi.org/10.1016/s0022-1759(98)00193-8

Publication

Journal of immunological methods
ISSN: 0022-1759
NlmUniqueID: 1305440
Country: Netherlands
Language: English
Volume: 222
Issue: 1-2
Pages: 155-169

Researcher Affiliations

Swiderski, C E
  • Department of Veterinary Microbiology and Parasitology, School of Veterinary Medicine, Louisiana State University, Baton Rouge 70803, USA.
Klei, T R
    Horohov, D W

      MeSH Terms

      • Actins / biosynthesis
      • Actins / genetics
      • Animals
      • DNA, Complementary / genetics
      • DNA, Complementary / metabolism
      • Gene Amplification
      • Horses
      • Interleukin-10 / biosynthesis
      • Interleukin-10 / genetics
      • Leukocytes, Mononuclear / metabolism
      • RNA, Messenger / blood
      • RNA, Messenger / genetics
      • Reference Standards
      • Reproducibility of Results
      • Reverse Transcriptase Polymerase Chain Reaction
      • Sensitivity and Specificity

      Citations

      This article has been cited 12 times.
      1. Attia MM, Soliman SM, Salaeh NMK, Salem HM, Alkafafy M, Saad AM, El-Saadony MT, El-Gameel SM. Evaluation of immune responses and oxidative stress in donkeys: Immunological studies provoked by Parascaris equorum infection.. Saudi J Biol Sci 2022 Apr;29(4):2173-2179.
        doi: 10.1016/j.sjbs.2021.11.044pubmed: 35531146google scholar: lookup
      2. Torres R, Hurtado C, Pérez-Macchi S, Bittencourt P, Freschi C, de Mello VVC, Machado RZ, André MR, Müller A. Occurrence and Genetic Diversity of Babesia caballi and Theileria equi in Chilean Thoroughbred Racing Horses.. Pathogens 2021 Jun 7;10(6).
        doi: 10.3390/pathogens10060714pubmed: 34200433google scholar: lookup
      3. Morini M, Peli A, Rinnovati R, Magazzù G, Romagnoli N, Spadari A, Pietra M. Immunohistochemical Expression of Neurokinin-A and Interleukin-8 in the Bronchial Epithelium of Horses with Severe Equine Asthma Syndrome during Asymptomatic, Exacerbation, and Remission Phase.. Animals (Basel) 2021 May 12;11(5).
        doi: 10.3390/ani11051376pubmed: 34066204google scholar: lookup
      4. Yamaoka H, Nishizawa S, Asawa Y, Fujihara Y, Ogasawara T, Yamaoka K, Nagata S, Takato T, Hoshi K. Involvement of fibroblast growth factor 18 in dedifferentiation of cultured human chondrocytes.. Cell Prolif 2010 Feb;43(1):67-76.
        doi: 10.1111/j.1365-2184.2009.00655.xpubmed: 19909293google scholar: lookup
      5. Allen CA, Payne SL, Harville M, Cohen N, Russell KE. Validation of quantitative polymerase chain reaction assays for measuring cytokine expression in equine macrophages.. J Immunol Methods 2007 Dec 1;328(1-2):59-69.
        doi: 10.1016/j.jim.2007.08.006pubmed: 17900609google scholar: lookup
      6. van den Hoven R, Duvigneau JC, Hartl RT, Gemeiner M. Clenbuterol affects the expression of messenger RNA for interleukin 10 in peripheral leukocytes from horses challenged intrabronchially with lipopolysaccharides.. Vet Res Commun 2006 Nov;30(8):921-8.
        doi: 10.1007/s11259-006-3383-4pubmed: 17139543google scholar: lookup
      7. Bogaert L, Van Poucke M, De Baere C, Peelman L, Gasthuys F, Martens A. Selection of a set of reliable reference genes for quantitative real-time PCR in normal equine skin and in equine sarcoids.. BMC Biotechnol 2006 Apr 27;6:24.
        doi: 10.1186/1472-6750-6-24pubmed: 16643647google scholar: lookup
      8. Venugopal CS, Polikepahad S, Holmes EP, Heuvel JV, Leas TL, Moore RM. Endothelin receptor alterations in equine airway hyperreactivity.. Can J Vet Res 2006 Jan;70(1):50-7.
        pubmed: 16548332
      9. Chirgwin SR, Coleman SU, Porthouse KH, Nowling JM, Punkosdy GA, Klei TR. Removal of Wolbachia from Brugia pahangi is closely linked to worm death and fecundity but does not result in altered lymphatic lesion formation in Mongolian gerbils (Meriones unguiculatus).. Infect Immun 2003 Dec;71(12):6986-94.
        doi: 10.1128/IAI.71.12.6986-6994.2003pubmed: 14638788google scholar: lookup
      10. Chirgwin SR, Elzer PH, Coleman SU, Nowling JM, Hagius SD, Edmonds MD, Klei TR. Infection outcome and cytokine gene expression in Brugia pahangi- infected gerbils (Meriones unguiculatus) sensitized with Brucella abortus.. Infect Immun 2002 Nov;70(11):5938-45.
        doi: 10.1128/IAI.70.11.5938-5945.2002pubmed: 12379668google scholar: lookup
      11. Klein D, Denis M, Ricordi C, Pastori RL. Assessment of ribozyme cleavage efficiency using reverse transcriptase real-time PCR.. Mol Biotechnol 2000 Mar;14(3):189-95.
        doi: 10.1385/MB:14:3:189pubmed: 10890009google scholar: lookup
      12. Giguère S, Wilkie BN, Prescott JF. Modulation of cytokine response of pneumonic foals by virulent Rhodococcus equi.. Infect Immun 1999 Oct;67(10):5041-7.
        doi: 10.1128/IAI.67.10.5041-5047.1999pubmed: 10496876google scholar: lookup
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