Standardization of preclinical methodologies for discovery and validation of circulating microRNA biomarkers for post-traumatic epileptogenesis – Lessons learned from the EpiBioS4Rx Project 1

OBJECTIVE: To analyze the success of harmonization and standardization of plasma miRNA biomarker discovery and validation for post-traumatic epilepsy (PTE) in the EpiBioS4Rx international multicenter project. METHODS: Adult male Sprague-Dawley rats were randomized to lateral fluid-percussion-induced traumatic brain injury (TBI) or sham operation at three study sites (Finland, Australia, USA). Video-electroencephalogram (vEEG) was performed in the 7th post-injury month to detect spontaneous seizures. Tail vein plasma was collected at baseline and 48 h after TBI for microRNA (miRNA) analysis. Common data elements were generated to document and monitor pre-analytic activities, including housing conditions, post-injury care, blood sampling, plasma preparation, plasma quality, storage, and shipping. miRNA analysis was performed using droplet digital PCR (ddPCR) at one study site (Finland) with on-site standardized procedures. RESULTS: The 2-day miRNA levels were successfully measured in 85 % (209/245) of the rats included in the final analysis cohort. Exclusions were related to small sample volume, hemolysis, and failed RNA extraction for ddPCR. Most of the pre-analytical factors leading to sample exclusions were related to non-optimal plasma pipetting. We also recognized gaps in data entry and monitoring of personnel training. CONCLUSIONS: Our study demonstrates that conducting a successful plasma miRNA biomarker analysis requires procedural harmonization between laboratories, protocol standardization, inclusion and analysis of quality controls, training of researchers, and continuous monitoring of adherence to pre-agreed protocols.