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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

References

This article includes 41 references
  1. Teale P, Barton C, Driver PM, Kay RG. Biomarkers: unrealized potential in sports doping analysis. Bioanalysis 2009;1(6):1103-1118.
    doi: 10.4155/bio.09.87google scholar: lookup
  2. Aronson JK, Ferner RE. Biomarkers-a general review. Curr Protoc Pharmacol 2017;76(1):9.23.1-9.23.17.
    doi: 10.1002/cpph.19google scholar: lookup
  3. Quezada H, Guzmán-Ortiz AL, Díaz-Sánchez H, Valle-Rios R, Aguirre-Hernández J. Omics-based biomarkers: current status and potential use in the clinic. Bol Med Hosp Infant Mex 2017;74(3):219-226.
    doi: 10.1016/j.bmhimx.2017.03.003google scholar: lookup
  4. He T. Implementation of proteomics in clinical trials. Proteomics Clin Appl 2019;13(2):1800198.
    doi: 10.1002/prca.201800198google scholar: lookup
  5. He YD. Genomic approach to biomarker identification and its recent applications. Cancer Biomark Sect Dis Markers 2006;2(3-4):103-133.
    doi: 10.3233/cbm-2006-23-404google scholar: lookup
  6. Monteiro, Carvalho M, Bastos ML, Guedes de Pinho P. Metabolomics analysis for biomarker discovery: advances and challenges. Curr Med Chem 2013;20(2):257-271.
    doi: 10.2174/092986713804806621google scholar: lookup
  7. López-Barea J. Biomarkers in ecotoxicology: an overview. Arch Toxicol Suppl Arch Toxikol Suppl 1995;17:57-79.
    doi: 10.1007/978-3-642-79451-3_6google scholar: lookup
  8. Sottas P-E, Robinson N, Rabin O, Saugy M. The athlete biological passport. Clin Chem 2011;57(7):969-976.
    doi: 10.1373/clinchem.2011.162271google scholar: lookup
  9. Duluard A, Bailly-Chouriberry L, Kieken F, Popot M-A, Bonnaire Y. Longitudinal follow-up on racehorses: veterinary and analytical issues. A one-year study of French trotters. Proc 18th Int Conf Racing Anal Vet ICRAV N Z 2010;27-34.
  10. Cawley AT, Keledjian J. Intelligence-based anti-doping from an equine biological passport. Drug Test Anal 2017;9(9):1441-1447.
    doi: 10.1002/dta.2180google scholar: lookup
  11. Bailly-Chouriberry L, Noguier F, Manchon L. Blood cells RNA biomarkers as a first long-term detection strategy for EPO abuse in horseracing. Drug Test Anal 2010;2(7):339-345.
    doi: 10.1002/dta.146google scholar: lookup
  12. Wong K-S, Cheung HW, Choi TLS. Label-free proteomics for discovering biomarker candidates for controlling krypton misuse in castrated horses (geldings). J Proteome Res 2020;19(3):1196-1208.
    doi: 10.1021/acs.jproteome.9b00724google scholar: lookup
  13. Kieken F, Pinel G, Antignac J-P. Generation and processing of urinary and plasmatic metabolomic fingerprints to reveal an illegal administration of recombinant equine growth hormone from LC-HRMS measurements. Metabolomics 2011;7(1):84-93.
    doi: 10.1007/s11306-010-0233-8google scholar: lookup
  14. Joré C, Loup B, Garcia P. Liquid chromatography-high resolution mass spectrometry-based metabolomic approach for the detection of continuous erythropoiesis receptor activator effects in horse doping control. J Chromatogr A 2017;1521:90-99.
    doi: 10.1016/j.chroma.2017.09.029google scholar: lookup
  15. Kaabia Z, Dervilly-Pinel G, Popot MA. Monitoring the endogenous steroid profile disruption in urine and blood upon nandrolone administration: an efficient and innovative strategy to screen for nandrolone abuse in entire male horses. Drug Test Analyst 2014;6(4):376-388.
    doi: 10.1002/dta.1520google scholar: lookup
  16. Chan GHM, Ho ENM, Leung DKK, Wong KS, Wan TSM. Targeted metabolomics approach to detect the misuse of steroidal aromatase inhibitors in equine sports by biomarker profiling. Anal Chem 2016;88(1):764-772.
    doi: 10.1021/acs.analchem.5b03165google scholar: lookup
  17. Neuberger EWI, Moser DA, Simon P. Principle considerations for the use of transcriptomics in doping research. Drug Test Anal 2011;3(10):668-675.
    doi: 10.1002/dta.331google scholar: lookup
  18. Varlet-Marie E, Audran M, Ashenden M, Sicart M-T, Piquemal D. Modification of gene expression: help to detect doping with erythropoiesis-stimulating agents. Am J Hematol 2009;84(11):755-759.
    doi: 10.1002/ajh.21525google scholar: lookup
  19. Reiter M, Pfaffl MW, Schonfelder M, Meyer HHD. Gene expression in hair follicle dermal papilla cells after treatment with stanozolol. Biomark Insights 2008;4:1-8.
  20. Riedmaier I, Pfaffl MW, Meyer HHD. The physiological way: monitoring RNA expression changes as new approach to combat illegal growth promoter application. Drug Test Anal 2012;4:70-74.
    doi: 10.1002/dta.1386google scholar: lookup
  21. Riedmaier I, Pfaffl MW, Meyer HHD. The analysis of the transcriptome as a new approach for biomarker development to trace the abuse of anabolic steroid hormones. Drug Test Anal 2011;3(10):676-681.
    doi: 10.1002/dta.304google scholar: lookup
  22. Mitchell CJ, Nelson AE, Cowley MJ. Detection of growth hormone doping by gene expression profiling of peripheral blood. J Clin Endocrinol Metab 2009;94(12):4703-4709.
    doi: 10.1210/jc.2009-1038google scholar: lookup
  23. Hollis AR, Starkey MP. MicroRNAs in equine veterinary science. Equine Vet J 2018;50(6):721-726.
    doi: 10.1111/evj.12954google scholar: lookup
  24. Leuenberger N, Jan N, Pradervand S, Robinson N, Saugy M. Circulating microRNAs as long-term biomarkers for the detection of erythropoiesis-stimulating agent abuse. Drug Test Anal 2011;3(11-12):771-776.
    doi: 10.1002/dta.370google scholar: lookup
  25. Salamin O, Jaggi L, Baume N, Robinson N, Saugy M, Leuenberger N. Circulating microRNA-122 as potential biomarker for detection of testosterone abuse. PLoS One 2016;11(5):e0155248.
    doi: 10.1371/journal.pone.0155248google scholar: lookup
  26. Kelly BN, Haverstick DM, Lee JK. Circulating microRNA as a biomarker of human growth hormone administration to patients. Drug Test Anal 2014;6(3):234-238.
    doi: 10.1002/dta.1469google scholar: lookup
  27. Leuenberger N, Schumacher YO, Pradervand S, Sander T, Saugy M, Pottgiesser T. Circulating microRNAs as biomarkers for detection of autologous blood transfusion. PLoS One 2013;8(6):e66309.
    doi: 10.1371/journal.pone.0066309google scholar: lookup
  28. Lee S, Hwang S, Yu HJ. Expression of microRNAs in horse plasma and their characteristic nucleotide composition. PLoS One 2016;11(1):e0146374.
    doi: 10.1371/journal.pone.0146374google scholar: lookup
  29. Pacholewska A, Mach N, Mata X. Novel equine tissue miRNAs and breed-related miRNA expressed in serum. BMC Genomics 2016;17(1):831.
    doi: 10.1186/s12864-016-3168-2google scholar: lookup
  30. Mach N, Plancade S, Pacholewska A. Integrated mRNA and miRNA expression profiling in blood reveals candidate biomarkers associated with endurance exercise in the horse. Sci Rep 2016;6(22932).
    doi: 10.1038/srep22932google scholar: lookup
  31. Lecchi C, Costa ED, Lebelt D. Circulating miR-23b-3p, miR-145-5p and miR-200b-3p are potential biomarkers to monitor acute pain associated with laminitis in horses. Animal 2018;12:366-375.
    doi: 10.1017/s1751731117001525google scholar: lookup
  32. Pawlina K, Gurgul A, Szmatoła T. Comprehensive characteristics of microRNA expression profile of equine sarcoids. Biochimie 2017;137:20-28.
    doi: 10.1016/j.biochi.2017.02.017google scholar: lookup
  33. Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright AJ. miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res 2006;34(90001):D140-D144.
    doi: 10.1093/nar/gkj112google scholar: lookup
  34. Beutler E, Gelbart T, Kuhl W. Interference of heparin with the polymerase chain reaction. Biotechniques 1990;9:166.
  35. Izraeli S, Pfleiderer C, Lion T. Detection of gene expression by PCR amplification of RNA derived from frozen heparinized whole blood. Nucleic Acids Res 1991;19(21):6051.
  36. Taylor AC. Titration of heparinase for removal of the PCR-inhibitory effect of heparin in DNA samples. Mol Ecol 1997;6(4):383-385.
    doi: 10.1046/j.1365-294x.1997.00191.xgoogle scholar: lookup
  37. Li S, Chen H, Song J, Lee C, Geng Q. Avoiding heparin inhibition in circulating MicroRNAs amplification. Int J Cardiol 2016;207:92-93.
    doi: 10.1016/j.ijcard.2016.01.129google scholar: lookup
  38. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215(3):403-410.
    doi: 10.1016/s0022-2836(05)80360-2google scholar: lookup
  39. Vandesompele J, De Preter K, Pattyn F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002;3:1-11.
  40. de Oliveira GP, Porto WF, Palu CC. Effects of endurance racing on horse plasma extracellular particle miRNA. Equine Vet J 2020;53(3):618-627.
    doi: 10.1111/evj.13300google scholar: lookup
  41. Carter EI, Valli VE, McSherry BJ, Lumsden JH, Milne FJ, Robinson GA. The kinetics of hematopoiesis in the light horse I. The lifespan of peripheral blood cells in the normal horse. Can J Comp Med 1974;38:303-313.

Citations

This article has been cited 5 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
  2. Postnikov PV, Pronina IV, Polyansky DS, Efimova YA, Ordzhonikidze ZG, Badtieva VA, Nikityuk DB. Analysis of Specific MicroRNAs Expressed after Intake of Cobalt and Xenon Preparations for Anti-Doping Control. Bull Exp Biol Med 2025 Apr;178(6):726-732.
    doi: 10.1007/s10517-025-06406-xpubmed: 40442471google scholar: lookup
  3. Ishii H, Shigematsu R, Takemoto S, Ishikawa Y, Mizobe F, Nomura M, Arima D, Kunii H, Yuasa R, Yamanaka T, Tanabe S, Nagata SI, Yamada M, Leung GN. Quantification of osilodrostat in horse urine using LC/ESI-HRMS to establish an elimination profile for doping control. Bioanalysis 2024;16(17-18):947-958.
    doi: 10.1080/17576180.2024.2385848pubmed: 39235065google scholar: lookup
  4. Loria F, Grabherr S, Kuuranne T, Leuenberger N. Use of RNA biomarkers in the antidoping field. Bioanalysis 2024;16(10):475-484.
    doi: 10.4155/bio-2023-0251pubmed: 38497758google scholar: lookup
  5. Kikuchi M, Ishige T, Minamijima Y, Hirota KI, Nagata SI, Tozaki T, Kakoi H, Ishiguro-Oonuma T, Kizaki K. Identification of Potential miRNA Biomarkers to Detect Hydrocortisone Administration in Horses. Int J Mol Sci 2023 Sep 25;24(19).
    doi: 10.3390/ijms241914515pubmed: 37833961google scholar: lookup
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