Skip to main content
Log in

Mass spectrometric detection of siRNA in plasma samples for doping control purposes

  • Original Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Small interfering ribonucleic acid (siRNA) molecules can effect the expression of any gene by inducing the degradation of mRNA. Therefore, these molecules can be of interest for illicit performance enhancement in sports by affecting different metabolic pathways. An example of an efficient performance-enhancing gene knockdown is the myostatin gene that regulates muscle growth. This study was carried out to provide a tool for the mass spectrometric detection of modified and unmodified siRNA from plasma samples. The oligonucleotides are purified by centrifugal filtration and the use of an miRNA purification kit, followed by flow-injection analysis using an Exactive mass spectrometer to yield the accurate masses of the sense and antisense strands. Although chromatography and sensitive mass spectrometric analysis of oligonucleotides are still challenging, a method was developed and validated that has adequate sensitivity (limit of detection 0.25–1 nmol mL−1) and performance (precision 11–21%, recovery 23–67%) for typical antisense oligonucleotides currently used in clinical studies.

A method for the mass spectrometric detection of siRNA molecules in doping control is described. siRNA, which blocks the translation of genes, could be used by athletes for illicit performance enhancement by e.g. down-regulating the myostatin gene for enhanced muscle growth.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Scheme 1
Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Whitehead KA, Langer R, Anderson DG (2009) Knocking down barriers: advances in siRNA delivery. Nat Rev Drug Discov 8:129–138

    Article  CAS  Google Scholar 

  2. Kurreck J (2009) RNA interference: from basic research to therapeutic applications. Angew Chem Int Ed Engl 48:1378–1398

    Article  CAS  Google Scholar 

  3. Rana TM (2007) Illuminating the silence: understanding the structure and function of small RNAs. Nat Rev Mol Cell Biol 8:23–36

    Article  CAS  Google Scholar 

  4. Corey DR (2007) Chemical modification: the key to clinical application of RNA interference? J Clin Invest 117:3615–3622

    Article  CAS  Google Scholar 

  5. Kurreck J (2003) Antisense technologies. Improvement through novel chemical modifications. Eur J Biochem 270:1628–1644

    Article  CAS  Google Scholar 

  6. Juliano R, Bauman J, Kang H, Ming X (2009) Biological barriers to therapy with antisense and siRNA oligonucleotides. Mol Pharm 6:686–695

    Article  CAS  Google Scholar 

  7. Bramsen JB, Laursen MB, Nielsen AF, Hansen TB, Bus C, Langkjaer N, Babu BR, Hojland T, Abramov M, Van Aerschot A et al (2009) A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity. Nucleic Acids Res 37:2867–2881

    Article  CAS  Google Scholar 

  8. Gao S, Dagnaes-Hansen F, Nielsen EJ, Wengel J, Besenbacher F, Howard KA, Kjems J (2009) The effect of chemical modification and nanoparticle formulation on stability and biodistribution of siRNA in mice. Mol Ther 17:1225–1233

    Article  CAS  Google Scholar 

  9. Cho IS, Kim J, Lim do H, Ahn HC, Kim H, Lee KB, Lee YS (2008) Improved serum stability and biophysical properties of siRNAs following chemical modifications. Biotechnol Lett 30:1901–1908

    Article  CAS  Google Scholar 

  10. Behlke MA (2008) Chemical modification of siRNAs for in vivo use. Oligonucleotides 18:305–319

    Article  CAS  Google Scholar 

  11. Bumcrot D, Manoharan M, Koteliansky V, Sah DW (2006) RNAi therapeutics: a potential new class of pharmaceutical drugs. Nat Chem Biol 2:711–719

    Article  CAS  Google Scholar 

  12. Haussecker D (2008) The business of RNAi therapeutics. Hum Gene Ther 19:451–462

    Article  CAS  Google Scholar 

  13. Schuelke M, Wagner KR, Stolz LE, Hubner C, Riebel T, Komen W, Braun T, Tobin JF, Lee SJ (2004) Myostatin mutation associated with gross muscle hypertrophy in a child. N Engl J Med 350:2682–2688

    Article  CAS  Google Scholar 

  14. Liu CM, Yang Z, Liu CW, Wang R, Tien P, Dale R, Sun LQ (2008) Myostatin antisense RNA-mediated muscle growth in normal and cancer cachexia mice. Gene Ther 15:155–160

    Article  Google Scholar 

  15. Acosta J, Carpio Y, Borroto I, Gonzalez O, Estrada MP (2005) Myostatin gene silenced by RNAi show a zebrafish giant phenotype. J Biotechnol 119:324–331

    Article  CAS  Google Scholar 

  16. Tsuchida K (2008) Targeting myostatin for therapies against muscle-wasting disorders. Curr Opin Drug Discov Dev 11:487–494

    CAS  Google Scholar 

  17. Huang TY, Liu J, Liang X, Hodges BD, McLuckey SA (2008) Collision-induced dissociation of intact duplex and single-stranded siRNA anions. Anal Chem 80:8501–8508

    Article  CAS  Google Scholar 

  18. Bahr U, Aygun H, Karas M (2008) Detection and relative quantification of siRNA double strands by MALDI mass spectrometry. Anal Chem 80:6280–6285

    Article  CAS  Google Scholar 

  19. Huber CG, Buchmeiser MR (1998) On-line cation exchange for suppression of adduct formation in negative-ion electrospray mass spectrometry of nucleic acids. Anal Chem 70:5288–5295

    Article  CAS  Google Scholar 

  20. Zou Y, Tiller P, Chen IW, Beverly M, Hochman J (2008) Metabolite identification of small interfering RNA duplex by high-resolution accurate mass spectrometry. Rapid Commun Mass Spectrom 22:1871–1881

    Article  CAS  Google Scholar 

  21. Zhang G, Lin J, Srinivasan K, Kavetskaia O, Duncan JN (2007) Strategies for bioanalysis of an oligonucleotide class macromolecule from rat plasma using liquid chromatography–tandem mass spectrometry. Anal Chem 79:3416–3424

    Article  CAS  Google Scholar 

  22. McCarthy SM, Gilar M, Gebler J (2009) Reversed-phase ion-pair liquid chromatography analysis and purification of small interfering RNA. Anal Biochem 390:181–188

    Article  CAS  Google Scholar 

  23. Thevis M, Schänzer W (2007) Current role of LC–MS(/MS) in doping control. Anal Bioanal Chem 388:1351–1358

    Article  CAS  Google Scholar 

  24. Thevis M, Schänzer W (2007) Mass spectrometry in sports drug testing: Structure characterization and analytical assays. Mass Spectrom Rev 26:79–107

    Article  CAS  Google Scholar 

  25. Greig M, Griffey RH (1995) Utility of organic bases for improved electrospray mass spectrometry of oligonucleotides. Rapid Commun Mass Spectrom 9:97–102

    Article  CAS  Google Scholar 

  26. Andreasen D, Fog JU, Biggs W, Salomon J, Dahslveen IK, Baker A, Mouritzen P (2010) Improved microRNA quantification in total RNA from clinical samples. Methods 50:S6–S9

    Article  CAS  Google Scholar 

  27. Wang K, Zhang S, Marzolf B, Troisch P, Brightman A, Hu Z, Hood LE, Galas DJ (2009) Circulating microRNAs, potential biomarkers for drug-induced liver injury. Proc Natl Acad Sci USA 106:4402–4407

    Article  CAS  Google Scholar 

  28. Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, Peterson A, Noteboom J, O'Briant KC, Allen A et al (2008) Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci USA 105:10513–10518

    Article  CAS  Google Scholar 

  29. Mandel J (1964) The statistical analyses of experimental data. John Wiley & Sons, New York

    Google Scholar 

Download references

Acknowledgements

The study was carried out with the support of Antidoping Switzerland (Berne, Switzerland), the Manfred Donike Institute of Doping Analysis, and the Federal Ministry of the Interior of the Federal Republic of Germany.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Mario Thevis.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kohler, M., Thomas, A., Walpurgis, K. et al. Mass spectrometric detection of siRNA in plasma samples for doping control purposes. Anal Bioanal Chem 398, 1305–1312 (2010). https://doi.org/10.1007/s00216-010-4013-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-010-4013-0

Keywords

Navigation