title: Optimized microRNA purification from TRIzol-treated plasma.
abstract: BACKGROUND
  MicroRNAs (miRNAs) represent new and potentially informative diagnostic targets for disease detection and prognosis. However, little work exists documenting the effect of TRIzol, a common viral inactivation and nucleic acid extraction reagent, on miRNA purification. Here, we developed an optimized protocol for miRNA extraction from plasma samples by evaluating five different RNA extraction kits, TRIzol phase separation, purification additives, and initial plasma sample volume. This method was then used for downstream profiling of plasma miRNAs found in archived samples from one nonhuman primate (NHP) experimentally challenged with Ebola virus by the aerosol route.
  
  
  RESULTS
  Comparison of real-time RT-PCR results for spiked-in and endogenous miRNA sequences determined extraction efficiencies from five different RNA purification kits. These experiments showed that 50 μL plasma processed using the QIAGEN miRNeasy Mini Kit with 5 μg of glycogen as a co-precipitant yielded the highest recovery of endogenous miRNAs. Using this optimized protocol, miRNAs from archived plasma samples of one rhesus macaque challenged with aerosolized Ebola virus was profiled using a targeted real-time PCR array. A total of 519 of the 752 unique miRNAs assayed were present in the plasma samples at day 0 and day 7 (time of death) post-exposure. Statistical analyses revealed 25 sequences significantly up- or down-regulated between day 0 and day 7 post infection, validating the utility of the extraction method for plasma miRNA profiling.
  
  
  CONCLUSIONS
  This study contributes to the knowledgebase of circulating miRNA extraction methods and expands on the potential applications of cell-free miRNA profiling for diagnostics and pathogenesis studies. Specifically, we optimized an extraction protocol for miRNAs from TRIzol-inactivated plasma samples that can be used for highly pathogenic viruses.
authors.affiliation: Diagnostic Systems Division, U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter St., Fort Detrick, Frederick, MD, 21701, USA. janice.p.duy.ctr@mail.mil.
authors.forename: Janice
authors.initials: J
authors.lastname: Duy
authors.affiliation: Diagnostic Systems Division, U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter St., Fort Detrick, Frederick, MD, 21701, USA. jeffrey.w.koheler.ctr@mail.mil.
authors.forename: Jeffrey W
authors.initials: JW
authors.lastname: Koehler
authors.affiliation: Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 8200 Research Plaza, Fort Detrick, Frederick, MD, 21701, USA. anna.honko@nih.gov.|Virology Division, U.S. Army Medical Institute of Infectious Diseases, 1425 Porter St., Fort Detrick, Frederick, MD, 21701, USA. anna.honko@nih.gov.
authors.forename: Anna N
authors.initials: AN
authors.lastname: Honko
authors.affiliation: Diagnostic Systems Division, U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter St., Fort Detrick, Frederick, MD, 21701, USA. timothy.d.minogue.civ@mail.mil.
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Minogue
mesh_terms: D000818:Animals; D029043:Ebolavirus / Q000235:genetics; D006146:Guanidines / Q000494:pharmacology; D019142:Hemorrhagic Fever, Ebola / Q000097:blood / Q000235:genetics* / Q000821:virology; D008253:Macaca mulatta / Q000097:blood / Q000235:genetics / Q000821:virology; D035683:MicroRNAs / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification*; D010636:Phenols / Q000494:pharmacology
chemical_list: D006146:Guanidines; D035683:MicroRNAs; D010636:Phenols; C411644:trizol