Epilepsy research · 2025
Standardization of preclinical methodologies for discovery and validation of circulating microRNA biomarkers for post-traumatic epileptogenesis - Lessons learned from the EpiBioS4Rx Project 1.
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Puhakka Noora, Heiskanen Mette, Ndode-Ekane Xavier Ekolle, Ali Idrish, Santana-Gomez Cesar, Das Gupta Shalini, Banerjee Meheli, Andrade Pedro, Immonen Riikka, Casillas-Espinosa Pablo, Smith Gregory, Brady Rhys D, Silva Juliana, Braine Emma, Hudson Matthew R, Yamakawa Glen R, Jones Nigel C, Shultz Sandy R, Harris Neil G, Wright David K, Gröhn Olli, Staba Richard, O'Brien Terence J, Pitkänen Asla
Аннотация
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.
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.