Universität zu Köln

Thomas, Andreas ORCID: 0000-0003-1199-0743, Walpurgis, Katja, Delahaut, Philippe, Kohler, Maxie, Schaenzer, Wilhelm and Thevis, Mario (2013). Detection of small interfering RNA (siRNA) by mass spectrometry procedures in doping controls. Drug Test. Anal., 5 (11-12). S. 853 - 861. HOBOKEN: WILEY-BLACKWELL. ISSN 1942-7611

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Abstract

Uncovering manipulation of athletic performance via small interfering (si)RNA is an emerging field in sports drug testing. Due to the potential to principally knock down every target gene in the organism by means of the RNA interference pathway, this facet of gene doping has become a realistic scenario. In the present study, two distinct model siRNAs comprising 21 nucleotides were designed as double strands which were perfect counterparts to a sequence of the respective messenger RNA coding the muscle regulator myostatin of Rattus norvegicus. Several modified nucleotides were introduced in both the sense and the antisense strand comprising phosphothioates, 2'-O-methylation, 2'-fluoro-nucleotides, locked nucleic acids and a cholesterol tag at the 3'-end. The model siRNAs were applied to rats at 1 mg/kg (i.v.) and blood as well as urine samples were collected. After isolation of the RNA by means of a RNA purification kit, the target analytes were detected by liquid chromatography - high resolution/high accuracy mass spectrometry (LC-HRMS). Analytes were detected as modified nucleotides after alkaline hydrolysis, as intact oligonucleotide strands (top-down) and by means of denaturing SDS-PAGE analysis. The gel-separated siRNA was further subjected to in-gel hydrolysis with different RNases and subsequent identification of the fragments by untargeted LC-HRMS analysis (bottom-up, experimental RNomics'). Combining the results of all approaches, the identification of several 3'-truncated urinary metabolites was accomplished and target analytes were detected up to 24 h after a single administration. Simultaneously collected blood samples yielded no promising results. The methods were validated and found fit-for-purpose for doping controls. Copyright (c) 2013 John Wiley & Sons, Ltd.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Thomas, Andreas UNSPECIFIED orcid.org/0000-0003-1199-0743 UNSPECIFIED Walpurgis, Katja UNSPECIFIED UNSPECIFIED UNSPECIFIED Delahaut, Philippe UNSPECIFIED UNSPECIFIED UNSPECIFIED Kohler, Maxie UNSPECIFIED UNSPECIFIED UNSPECIFIED Schaenzer, Wilhelm UNSPECIFIED UNSPECIFIED UNSPECIFIED Thevis, Mario UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-472324
DOI:	10.1002/dta.1519
Journal or Publication Title:	Drug Test. Anal.
Volume:	5
Number:	11-12
Page Range:	S. 853 - 861
Date:	2013
Publisher:	WILEY-BLACKWELL
Place of Publication:	HOBOKEN
ISSN:	1942-7611
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language PERFORMANCE LIQUID-CHROMATOGRAPHY; PHOSPHOROTHIOATE OLIGONUCLEOTIDE; THERAPEUTIC OLIGONUCLEOTIDES; ANTISENSE; BARRIERS; DELIVERY; PLASMA Multiple languages Biochemical Research Methods; Chemistry, Analytical; Pharmacology & Pharmacy Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/47232