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Updated: Jul 1, 2026
Isolation of Small Noncoding RNAs from Human Serum
Published on: June 19, 2014
Optimization of miRNA Serum Extraction: Processes and Lessons Learned in Preparation for Archival Serum Extractions
Andrew J Hoisington1,2,3, Christopher E Stamper1, Anna C Rivera4,5
1Veterans Health Administration, Rocky Mountain Mental Illness Research Education and Clinical Center (MIRECC) for Veteran Suicide Prevention, Rocky Mountain Regional Veterans Affairs Medical Center, Aurora, CO 80045, United States.
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.
Insights
This study optimized microRNA (miRNA) extraction from aged military serum samples, enabling successful RNA sequencing for over 86% of samples. The new protocols aid in identifying early disease biomarkers and understanding toxic exposures.
Area of Science:
- Biomarker Discovery
- Epigenetics
- Occupational Health
Background:
- Military personnel face airborne hazards, potentially causing adverse health outcomes via biological mechanisms.
- Alterations in microRNA (miRNA) expression may indicate epigenetic effects of environmental or occupational exposures.
- The Department of Defense Serum Repository (DODSR) stores decades-old serum samples not ideal for RNA preservation.
Purpose of the Study:
- To develop optimized miRNA extraction protocols for RNA sequencing of aged DODSR samples.
- To establish a proof of concept for processing and sequencing over 1,000 DODSR samples.
- To provide protocols applicable to other biorepositories for analyzing stored samples.
Main Methods:
- Seven optimization experiments were performed on serum samples stored for up to 2 years.
- A total of 1,016 DODSR samples were processed using the optimized method.
- miRNA extraction and quality control were assessed via TapeStation or paired-end sequencing.
Main Results:
- Successful miRNA sequencing was achieved for over 86% of samples, with an average collection age of over 16 years.
- A significant correlation was found between sample age and successful sequencing.
- Essential optimization factors included spike RNA incorporation and controlled sample thawing using an aluminum block.
Conclusions:
- Protocols were established for measuring miRNA in DODSR samples.
- These protocols support research into early detection biomarkers.
- The findings facilitate studies on mechanistic links between occupational exposures and adverse health outcomes.
