RNA-sequencing as useful screening tool in the combat against the misuse of anabolic agents

The abuse of anabolic substances in animal husbandry is forbidden within the EU and well controlled by detecting substance residues in different matrices. The application of newly designed drugs or substance cocktails represents big problems. Therefore developing sensitive test methods is important. The analysis of physiological changes caused by the use of anabolic agents on the molecular level, for example, by quantifying gene expression response, is a new approach to develop such screening methods. A novel technology for holistic gene expression analysis is RNA sequencing. In this study, the potential of this high-throughput method for the identification of biomarkers was evaluated. The effect of trenbolone acetate plus estradiol on gene expression in liver from Nguni heifers was analyzed with RNA sequencing. The expression of 40 selected candidate genes was verified via RT-qPCR, whereby 20 of these genes were significantly regulated. To extract the intended information from these regulated genes, biostatistical tools for pattern recognition were applied and resulted in a clear separation of the treatment groups. Those candidate genes could be verified in boars and in calves treated with anabolic substances. These results show the potential of RNA sequencing to screen for biomarker candidates to detect the abuse of anabolics. The verification of these biomarkers in boars and calves leads to the assumption that gene expression biomarkers are independent of breed or even species and that biomarkers, identified in farm animals could also act as potential biomarker candidates to detect the abuse of anabolic substances in human sports.