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Drug testing and analysis2021; 14(5); 953-962; doi: 10.1002/dta.3047

miRNAs detection in equine plasma by quantitative polymerase chain reaction for doping control: Assessment of blood sampling and study of eca-miR-144 as potential erythropoiesis stimulating agent biomarker.

Abstract: Short half-life doping substances are, quickly eliminated and therefore difficult to control with traditional analytical chemistry methods. Indirect methods targeting biomarkers constitute an alternative to extend detection time frames in doping control analyses. Gene expression analysis (i.e., transcriptomics) has already shown interesting results in both humans and equines for erythropoietin (EPO), growth hormone (GH), and anabolic androgenic steroid (AAS) misuses. In humans, circulating cell-free microRNAs in plasma were described as new potential biomarkers for control of major doping agent (MDA) abuses. The development of a quantitative polymerase chain reaction (qPCR) method allowing the detection of circulating miRNAs was carried out on equine plasma collected on different type of tubes (EDTA, lithium-heparin [LiHep]). Although analyzing plasma collected in EDTA tubes is a standard method in molecular biology, analyzing plasma collected in LiHep tubes is challenging, as heparin is a reverse transcription (RT) and a PCR inhibitor. Different strategies were considered, and attention was paid on both miRNAs extraction quality and detection sensitivity. The detection of endogenous circulating miRNAs was performed and compared between the different types of tubes. In parallel, homologs of human miRNAs characterized as potential biomarkers of doping were sought in equine databases. The miRNA eca-miR-144, described as potential erythropoiesis stimulating agents (ESAs) administration candidate biomarker was retained and assessed in equine post-administration samples. The results about the qPCR method development and optimization are exposed as well as the equine miRNAs detection. To our knowledge, this work is the first study and the proof of concept of circulating miRNAs detection in plasma dedicated to equine doping control.
© 2021 John Wiley & Sons, Ltd.
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Publication Date: 2021-05-03 PubMed ID: 33860991DOI: 10.1002/dta.3047Google Scholar: Lookup
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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.

The research article focuses on the detection of microRNAs in horse plasma through a method known as quantitative polymerase chain reaction (qPCR) in order to regulate doping in horse racing. It particularly looks at the possibility of using the eca-miR-144 microRNA as a biomarker for doping agents that stimulate erythropoiesis (red blood cell production).

Background and Need for the Study

  • Controlling the use of doping substances, particularly those with short half-lives that are quickly eliminated from the body, is challenging with conventional chemistry methods.
  • Biomarkers, substances used as indicators of a biological state or condition, can extend the detection timeframe.
  • MicroRNAs (small molecules that regulate gene expression) have been suggested as possible biomarkers for misuse of doping substances like erythropoietin (EPO), growth hormone (GH), and anabolic androgenic steroids (AAS).
  • No past anatomical studies have specifically focused on detecting circulating microRNAs for controlling doping in horses.

Method and Process

  • A quantitative polymerase chain reaction (qPCR) method was developed to detect circulating microRNAs in horse plasma.
  • Evaluation of this approach was performed using plasma collected in both EDTA and Lithium-Heparin (LiHep) tubes to ascertain the best collection tube for RNA extraction.
  • Although the use of EDTA tubes is common in molecular biology, the use of LiHep tubes presents a challenge as heparin inhibits reverse transcription and the PCR process.
  • The quality of extracted microRNAs and the sensitivity of their detection were also considered as part of the evaluation.
  • The detection of endogenous circulating microRNAs was performed and compared between the different tube types.

Identification of Potential Biomarkers

  • Equine homologs of human microRNAs that have been identified as possible doping biomarkers were searched for in equine databases.
  • The microRNA eca-miR-144 was selected as a promising biomarker for the misuse of erythropoiesis-stimulating agents (ESAs), substances that increase the production of red blood cells.
  • The potential of this microRNA was further evaluated using post-administration samples.

Results and Conclusion

  • The paper presents the results from the development and optimization of the qPCR method as well as the detection of equine microRNAs.
  • This study, being the first of its kind to look into the detection of circulating microRNAs in plasma for equine doping control, establishes a new way of detecting and controlling doping in horse racing.

Cite This Article

APA
Loup B, André F, Avignon J, Lhuaire M, Delcourt V, Barnabé A, Garcia P, Popot MA, Bailly-Chouriberry L. (2021). miRNAs detection in equine plasma by quantitative polymerase chain reaction for doping control: Assessment of blood sampling and study of eca-miR-144 as potential erythropoiesis stimulating agent biomarker. Drug Test Anal, 14(5), 953-962. https://doi.org/10.1002/dta.3047

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English
Volume: 14
Issue: 5
Pages: 953-962

Researcher Affiliations

Loup, Benoit
  • GIE Laboratoire des Courses Hippiques (LCH), 15 rue de Paradis, Verrières le Buisson, 91300, France.
André, François
  • GIE Laboratoire des Courses Hippiques (LCH), 15 rue de Paradis, Verrières le Buisson, 91300, France.
Avignon, Justine
  • GIE Laboratoire des Courses Hippiques (LCH), 15 rue de Paradis, Verrières le Buisson, 91300, France.
Lhuaire, Marion
  • GIE Laboratoire des Courses Hippiques (LCH), 15 rue de Paradis, Verrières le Buisson, 91300, France.
Delcourt, Vivian
  • GIE Laboratoire des Courses Hippiques (LCH), 15 rue de Paradis, Verrières le Buisson, 91300, France.
Barnabé, Agnès
  • GIE Laboratoire des Courses Hippiques (LCH), 15 rue de Paradis, Verrières le Buisson, 91300, France.
Garcia, Patrice
  • GIE Laboratoire des Courses Hippiques (LCH), 15 rue de Paradis, Verrières le Buisson, 91300, France.
Popot, Marie-Agnès
  • GIE Laboratoire des Courses Hippiques (LCH), 15 rue de Paradis, Verrières le Buisson, 91300, France.
Bailly-Chouriberry, Ludovic
  • GIE Laboratoire des Courses Hippiques (LCH), 15 rue de Paradis, Verrières le Buisson, 91300, France.

MeSH Terms

  • Animals
  • Biomarkers
  • Edetic Acid
  • Hematinics
  • Heparin
  • Horses / genetics
  • MicroRNAs
  • Polymerase Chain Reaction
  • Real-Time Polymerase Chain Reaction / methods

Grant Funding

  • Institut Franu00e7ais du Cheval et de l'Equitation

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Citations

This article has been cited 1 times.
  1. Dahlgren AR, Knych HK, Arthur RM, Durbin-Johnson BP, Finno CJ. Transcriptomic Markers of Recombinant Human Erythropoietin Micro-Dosing in Thoroughbred Horses.. Genes (Basel) 2021 Nov 24;12(12).
    doi: 10.3390/genes12121874pubmed: 34946824google scholar: lookup
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