Mass spectrometric detection of siRNA in plasma samples for doping control purposes
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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.
KeywordsDoping Sport Exactive mass spectrometry Oligonucleotide Duplex
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.
- 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 37:2867–2881CrossRefGoogle Scholar
- 16.Tsuchida K (2008) Targeting myostatin for therapies against muscle-wasting disorders. Curr Opin Drug Discov Dev 11:487–494Google Scholar
- 29.Mandel J (1964) The statistical analyses of experimental data. John Wiley & Sons, New YorkGoogle Scholar