Optimization of miRNA Serum Extraction: Processes and Lessons Learned in Preparation for Archival Serum Extractions.

INTRODUCTION: Military personnel are exposed to airborne hazards, such as jet fuel, that may lead to adverse health outcomes that come from biological mechanisms. In the serum of healthy individuals, the levels of microRNA are fairly consistent over time; therefore, alterations in the expression levels of specific miRNA may reflect the effects of environmental or occupational exposures on epigenetic mechanisms. The United States established a biorepository for storing serum samples collected from military members over the lifetime of their active duty (Department of Defense Serum Repository [DODSR]); however, samples may be decades old and are not stored in tubes meant to preserve RNA. The purpose of this study was to develop optimized miRNA extraction protocols for downstream RNA sequencing using DODSR samples as a proof of concept, with potential future applicability to other biorepositories. In addition, the optimized protocol was used to process and sequence over 1,000 DODSR samples.

MATERIALS AND METHODS: Seven optimization experiments were conducted using existing serum samples that were collected and frozen at -80 °C for up to 2 years prior to experiments. After optimizing the processing method, a total of 1,016 samples from the DODSR were processed as proof of concept. miRNAs were extracted, and quality control measures were assessed via the TapeStation system or paired-end sequencing.

RESULTS: We achieved successful miRNA sequencing for over 86% of samples that were, on average, collected more than 16 years prior. Furthermore, our analysis revealed a significant correlation between the ability to sequence serum samples and the age of the sample. Results from optimizing seven different processes suggested that incorporating spike RNA and tightly controlled sample thaws using an aluminum block are essential for high-quality sequence outcomes. Low concentration of miRNA in stored serum presents unique challenges for using standard quality control metrics.

CONCLUSIONS: Overall, this study established protocols for measuring miRNA in DODSR samples for studies that aim to elucidate early detection biomarkers or mechanistic associations between occupation-related toxic exposures and negative health outcomes.