Small RNA-Seq has emerged as a powerful tool in transcriptomics, gene expression profiling and biomarker discovery. Sequencing cell-free nucleic acids, particularly microRNA (miRNA), from liquid biopsies additionally provides exciting possibilities for molecular diagnostics, and might help establish disease-specific biomarker signatures. The complexity of the small RNA-Seq workflow, however, bears challenges and biases that researchers need to be aware of in order to generate high-quality data. Rigorous standardization and extensive validation are required to guarantee reliability, reproducibility and comparability of research findings. Hypotheses based on flawed experimental conditions can be inconsistent and even misleading. Comparable to the well-established MIQE guidelines for qPCR experiments, this work aims at establishing guidelines for experimental design and pre-analytical sample processing, standardization of library preparation and sequencing reactions, as well as facilitating data analysis. We highlight bottlenecks in small RNA-Seq experiments, point out the importance of stringent quality control and validation, and provide a primer for differential expression analysis and biomarker discovery. Following our recommendations will encourage better sequencing practice, increase experimental transparency and lead to more reproducible small RNA-Seq results. This will ultimately enhance the validity of biomarker signatures, and allow reliable and robust clinical predictions.

ObjectivesFor several decades, autologous blood doping (ABD) in sports has been a major problem, and even today there is still no reliable method for satisfactorily detecting ABD. For this kind of doping, stored individual erythrocytes are used to increase stamina and endurance caused by a higher erythrocyte level in the athlete’s body. Since there is growing evidence that these cells are enriched with microRNAs (miRNAs), this study has been carried out to discover and validate all miRNAs occurring in fresh blood as well as in stored blood.MethodsTherefore, small RNA Next Generation Sequencing has been performed, which allows untargeted detection of all miRNAs in a blood sample. The focus of this investigation has been to find miRNA alterations in blood bags after erythrocyte processing and during storage, as compared with fresh blood directly withdrawn from subjects. Blood samples were obtained from 12 healthy, recreationally active male subjects three times before blood donation and from blood bags at several time points after blood processing.Results189 miRNAs have been considered stable over two consecutive weeks. A further analysis revealed a complex biomarker signature of 28 miRNAs, consisting of 6 miRNAs that altered during 6 weeks of storage and 22 miRNAs that altered due to processing.ConclusionThese results suggest that the identified miRNA biomarker signature may be used for the detection of ABD. These 28 miRNA candidates are tested and verified currently in a follow-up study, a human transfusion clinical trial in healthy sportsmen.