title: Optimizing preservation of extracellular vesicular miRNAs derived from clinical cerebrospinal fluid.
abstract: BACKGROUND
  Tumor specific genetic material can be detected in extracellular vesicles (EVs) isolated from blood, cerebrospinal fluid (CSF), and other biofluids of glioblastoma patients. As such, EVs have emerged as a promising platform for biomarker discovery. However, the optimal procedure to transport clinical EV samples remains poorly characterized.
  
  
  METHODS
  We examined the stability of EVs isolated from CSF of glioblastoma patients that were stored under different conditions. EV recovery was determined by Nanoparticle tracking analysis, and qRT-PCR was performed to determine the levels of miRNAs.
  
  
  RESULTS
  CSF EVs that were lyophilized and stored at room temperature (RT) for seven days exhibited a 37-43% reduction in EV number. This reduction was further associated with decreased abundance of representative miRNAs. In contrast, the EV number and morphology remained largely unchanged if CSF were stored at RT. Total RNA and representative miRNA levels were well-preserved under this condition for up to seven days. A single cycle of freezing and thawing did not significantly alter EV number, morphology, RNA content, or miRNA levels. However, incremental decreases in these parameters were observed after two cycles of freezing and thawing.
  
  
  CONCLUSIONS
  These results suggest that EVs in CSF are stable at RT for at least seven days. Repeated cycles of freezing/thawing should be avoided to minimize experimental artifacts.
authors.affiliation: Center for Theoretical and Applied Neuro-Oncology, University of California, San Diego, CA, USA.
authors.forename: Johnny C
authors.initials: JC
authors.lastname: Akers
authors.affiliation: Center for Theoretical and Applied Neuro-Oncology, University of California, San Diego, CA, USA.
authors.forename: Valya
authors.initials: V
authors.lastname: Ramakrishnan
authors.affiliation: Department of Neurosurgery, University of California, Los Angeles, CA, USA.
authors.forename: Isaac
authors.initials: I
authors.lastname: Yang
authors.affiliation: Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Hua
authors.affiliation: Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Mao
authors.affiliation: Center for Theoretical and Applied Neuro-Oncology, University of California, San Diego, CA, USA.|Center for Theoretical and Applied Neuro-Oncology, University of California, San Diego, CA, USA.
authors.forename: Bob S
authors.initials: BS
authors.lastname: Carter
authors.affiliation: Center for Theoretical and Applied Neuro-Oncology, University of California, San Diego, CA, USA.|Center for Theoretical and Applied Neuro-Oncology, University of California, San Diego, CA, USA.
authors.forename: Clark C
authors.initials: CC
authors.lastname: Chen
mesh_terms: D015925:Cryopreservation; D000067128:Extracellular Vesicles / Q000235:genetics* / Q000648:ultrastructure; D018628:Gene Dosage; D006801:Humans; D035683:MicroRNAs / Q000134:cerebrospinal fluid / Q000235:genetics*; D020871:RNA Stability
chemical_list: D035683:MicroRNAs
keywords: CSF; EV; exosome; freeze thaw; lyophilization; stability