Validation of a reliable set of primer pairs for measuring gene expression by real-time quantitative RT-PCR in equine leukocytes.
Abstract: Quantification of gene expression using real-time reverse transcription quantitative PCR (RT-qPCR) is a reliable method to monitor cellular responses to pro-inflammatory stimuli. The main objective of this study was to validate a set of equine primer pairs that can be routinely used to monitor expression of genes that are central to inflammatory and immune responses. This paper describes the steps used to optimize and validate 29 equine primer pairs for RT-qPCR assays using SYBR Green detection. To validate these assays, monocytes were isolated from three horses and stimulated with Escherichia coli LPS. Because four of the 29 genes demonstrated poor amplification efficiency due to weak induction of gene expression by LPS, monocytes were stimulated with alternative agents (PMA and Poly I:C) known to induce gene expression in monocytes. These agents, acting through different pathways than LPS, improved the level of gene expression and yielded good amplification efficiency for these genes. PCR efficiency was based on a standard curve for each gene and the calculated efficiency was approximately 100% for all 29 genes. The PCR efficiencies for the majority of the target genes were equivalent to that of the housekeeping gene (18S rRNA) used in all experiments. Dissociation curve analysis and gel electrophoresis revealed a single product for each gene analyzed. To exemplify utilization of the validated primer pairs in studies of inflammatory cell activation, temporal changes in mRNA expression of a subset of genes were monitored in equine monocytes incubated with LPS. The availability of the 29 validated primer pairs reported herein will allow investigators to elucidate the response of horses to a variety of inflammatory stimuli and will further our understanding of disease pathogenesis in horses.
Publication Date: 2009-03-27 PubMed ID: 19376596DOI: 10.1016/j.vetimm.2009.03.013Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research is about the successful validation of a set of primer pairs for gene expression quantification in equine (horse) leukemia cells, which can provide insights into the horses’ response to inflammatory stimuli, improving the understanding of disease processes in these animals.
Overview of the Research
- The article discusses a study based on the quantification of gene expression using a real-time reverse transcription quantitative PCR (RT-qPCR). RT-qPCR is a method used reliably to monitor cellular responses, primarily to pro-inflammatory stimuli, which helps to understand the behavior of immune responses.
- As the main objective, the research aimed to confirm the reliability of a set of primer pairs specifically for equine or horse genes related to immune responses and inflammation. Primers are short pieces of DNA that are essential for PCR (Polymerase Chain Reaction) which is a common method to create multiple copies of a particular gene.
Validation Process
- The research involved the steps used to optimize and validate these 29 equine primer pairs for RT-qPCR assays using SYBR Green detection. SYBR Green detection is a widely used method for real-time PCR as it binds to any double-strand DNA sequence making it useful to understand and analyze the specifics of gene expression.
- In the validation process, monocytes (a type of white blood cell) were isolated from three horses and stimulated with Escherichia coli Lipopolysaccharides (LPS). LPS is a molecule present in the outer membrane of certain bacteria, like E.coli, and is known to trigger strong immune responses in animals.
The Results
- From these 29 genes, four showed poor amplification efficiency because of weak induction of gene expression by LPS. To fix this, they used alternative agents known to induce gene expression in monocytes, improving the level of gene expression which in turn improved the amplification efficiency for these genes.
- The article states that the PCR efficiency, calculated against a standard curve for each gene, was found to be approximately 100% for all 29 genes, which confirms the reliability of these primer pairs. The PCR efficiencies were equivalent to the housekeeping gene (18S rRNA) used as a control in all experiments.
- A single product was detected for each gene analyzed, affirming the specificity of the primer pairs.
- The validated primer pairs were used to monitor changes over time in mRNA expression of a selection of genes in equine monocytes treated with LPS, demonstrating their practical application.
Significance of the Study
- The outcomes of this study are beneficial as validated primer pairs are now available for studies focusing on inflammatory cell activation in horses. It will enable further investigations in the horse’s response to various inflammatory triggers, ultimately contributing to a more comprehensive understanding of disease development pathways in horses.
Cite This Article
APA
Figueiredo MD, Salter CE, Andrietti AL, Vandenplas ML, Hurley DJ, Moore JN.
(2009).
Validation of a reliable set of primer pairs for measuring gene expression by real-time quantitative RT-PCR in equine leukocytes.
Vet Immunol Immunopathol, 131(1-2), 65-72.
https://doi.org/10.1016/j.vetimm.2009.03.013 Publication
Researcher Affiliations
- Department of Large Animal Medicine, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602-7385, USA.
MeSH Terms
- Animals
- DNA Primers
- Horses / immunology
- Leukocytes / metabolism
- RNA, Messenger / analysis
- Reverse Transcriptase Polymerase Chain Reaction / methods
Citations
This article has been cited 18 times.- Seo MG, Ouh IO, Kwak D. Detection and Genotypic Analysis of Anaplasma bovis and A. phagocytophilum in Horse Blood and Lung Tissue. Int J Mol Sci 2023 Feb 7;24(4).
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- Selvarajah GT, Bonestroo FAS, Timmermans Sprang EPM, Kirpensteijn J, Mol JA. Reference gene validation for gene expression normalization in canine osteosarcoma: a geNorm algorithm approach. BMC Vet Res 2017 Nov 25;13(1):354.
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- Creamer DG, Schmiedt CW, Bullington AC, Caster CM, Schmiedt JM, Hurley DJ, Berghaus RD. Influence of exposure to microbial ligands, immunosuppressive drugs and chronic kidney disease on endogenous immunomodulatory gene expression in feline adipose-derived mesenchymal stem cells. J Feline Med Surg 2022 Jun;24(6):e43-e56.
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