Human plasma and serum extracellular small RNA reference profiles and their clinical utility

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 115; no. 23; pp. E5334 - E5343
• Main Authors: Max, Klaas E. A., Bertram, Karl, Akat, Kemal Marc, Bogardus, Kimberly A., Li, Jenny, Morozov, Pavel, Ben-Dov, Iddo Z., Li, Xin, Weiss, Zachary R., Azizian, Azadeh, Sopeyin, Anuoluwapo, Diacovo, Thomas G., Adamidi, Catherine, Williams, Zev, Tuschl, Thomas
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 05-06-2018
• Series: PNAS Plus
• Subjects: Adult; Biological Sciences; Biomarkers; Biomarkers - blood; Blood plasma; Cell-Free Nucleic Acids - blood; Cell-Free Nucleic Acids - genetics; Cell-Free Nucleic Acids - isolation & purification; Erythrocytes; Female; Females; Gene Library; Gene sequencing; Genetics; High-Throughput Nucleotide Sequencing; Humans; Male; MicroRNAs - blood; MicroRNAs - genetics; miRNA; Muscles; Phenotypes; Plasma; PNAS Plus; Proteolysis; Reducing agents; Ribonucleic acid; RNA; Standardization; exRNA biomarker; exRNA reference profiling; extracellular nucleic acids; biofluid DNA isolation; biofluid RNA isolation
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1714397115

Circulating extracellular RNAs (exRNAs) have the potential to serve as biomarkers for a wide range of medical conditions. However, limitations in existing exRNA isolation methods and a lack of knowledge on parameters affecting exRNA variability in human samples may hinder their successful discovery and clinical implementation. Using combinations of denaturants, reducing agents, proteolysis, and revised organic extraction, we developed an automated, high-throughput approach for recovery of exRNAs and exDNA from the same biofluid sample. We applied this method to characterize exRNAs from 312 plasma and serum samples collected from 13 healthy volunteers at 12 time points over a 2-month period. Small RNA cDNA library sequencing identified nearly twofold increased epithelial-, muscle-, and neuroendocrine-cell–specific miRNAs in females, while fasting and hormonal cycle showed little effect. External standardization helped to detect quantitative differences in erythrocyte and platelet-specific miRNA contributions and in miRNA concentrations between biofluids. It also helped to identify a study participant with a unique exRNA phenotype featuring a miRNA signature of up to 20-fold elevated endocrine-cell–specific miRNAs and twofold elevated total miRNA concentrations stable for over 1 year. Collectively, these results demonstrate an efficient and quantitative method to discern exRNA phenotypes and suggest that plasma and serum RNA profiles are stable over months and can be routinely monitored in long-term clinical studies.