Optimizing preservation of extracellular vesicular miRNAs derived from clinical cerebrospinal fluid

Johnny C. Akers, Valya Ramakrishnan, Isaac Yang, Wei Hua, Ying Mao, Bob S. Carter, Clark C. Chen

Research output: Contribution to journalArticlepeer-review

35 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)125-132
Number of pages8
JournalCancer Biomarkers
Volume17
Issue number2
DOIs
StatePublished - 2016

Bibliographical note

Funding Information:
Acknowledgments The work is supported by NIH UH2 TR000931-0, NIH PO1 2P30CA023100-28. Additional funding also provided by the Doris Duke Charitable Foundation Clinical Scientist Development Award, Sontag Foundation Distinguished Scientist Award, Burroughs Wellcome Fund Career Awards for Medical Scientists, the Kimmel Scholar Award, a Grant from Accelerated Brain Cancer Cure, the William Guy Forbeck Research Foundation, and Program of International Science & Technology Cooperation of China (2014DFA31470).

Keywords

  • CSF
  • EV
  • Exosome
  • Freeze thaw
  • Lyophilization
  • Stability

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