Development of microRNA based protocols for detection of autologous blood transfusion in sport

The implementation of the Athlete Biological Passport has increased the search for new biomarkers or techniques to detect Autologous Blood Transfusion (ABT). Genomics applied to microRNAs (miRNAs) is considered one of the most promising tools. Hypoxic and hyperoxic stimuli may influence miRNAs expression with changes potentially relevant for ABT detection. In a study recently completed and funded by the World Anti-Doping Agency (MARATHON), we have tested this hypothesis by searching for novel erythroid-specific miRNAs in a group of trained healthy volunteers exposed to ABT with respect to controls. The first result was the production of a validated WADA-UNIFE-Biobank constituted of around 2,000 plasma samples from control and transfused subjects. It is established that blood manipulation during withdrawal and reinfusion procedures might induce unpredictable changes of miRNAs content. Accordingly, a limited pool of samples derived from the cited Biobank was spontaneously tested in our laboratory for a global miRNA analysis, allowing the identification of a pool of erythroid associated miRNAs with changed plasma content in transfused subjects only. The primary objective of this project is to extend this analysis on the global miRNA content and identify changes of miRNAs (also in association with specific procedures of blood manipulation) of putative interest in ABT detection, taking advantage of the already available WADA-UNIFE Biobank. This experimental plan will allow to determine what is the best approach for obtaining a ABT-associated miRNA signature among the most important molecular biology approaches, including (a) quantitative RT-qPCR analysis on a limited set of validated erythroid-associated miRNAs; (b) same technical strategy on a set of miRNAs identified from a global microarray-based screening; (c) a microarray based clustering determining in the analyzed samples possible miRNA patterns similar to those obtained from ABT-trained samples