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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.
Publication Date: 1999-02-18 PubMed ID: 10022382DOI: 10.1016/s0022-1759(98)00193-8Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- 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
Researcher Affiliations
- Department of Veterinary Microbiology and Parasitology, School of Veterinary Medicine, Louisiana State University, Baton Rouge 70803, USA.
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 14 times.- 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.
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- 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).
- 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.
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- Suntrarachun S, Laoungbua P, Khunsap S, Noiporm J, Suttisee R. Evaluation of cellular immune response in rabbits after exposure to cobra venom and purified toxin fraction. Environ Anal Health Toxicol 2024 Dec;39(4):e2024029-0.
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