Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Optimizing preservation of extracellular vesicular miRNAs derived from clinical cerebrospinal fluid.

Author(s): Akers JC, Ramakrishnan V, Yang I, Hua W, Mao Y, Carter BS, Chen CC

Publication: Cancer Biomark, 2016, Vol. 17, Page 125-32

PubMed ID: 27062568 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare EV recovery, morphology, size distribution, and miRNA levels between CSF stored for 1 d or 7 days at room temperature; between EVs stored frozen and those lyophilized and stored at room temperature; and among isolated EVs subjected to up to three freeze-thaw cycles.  CSF was collected by ventricular/lumbar drain or aspiration during craniotomy from three glioblastoma patients. CSF was filtered through a 0.8 µm filter and stored for 0, 1, or 7 days at room temperature before frozen storage at -80°C. EVs were isolated from thawed CSF by differential centrifugation at 4°C (2,000 g for 20 min followed by 120,000 g for 2 h), resuspended in PBS and stored at -80°C.  Matched EV aliquots were stored frozen or lyophilized using a FreeZone Benchtop Freeze Dry system for 24 h and stored at room temperature for 7 days before rehydration in distilled water on ice. Additional EV aliquots were subjected to 0, 1, 2, and 3 freeze-thaw cycles. EVs were quantified by nanoparticle tracking analysis on a on a Nanosight LM-10HS machine and morphology was evaluated by transmission electron microscopy. RNA was isolated from EVs using the Qiagen miRNeasy Mini Kit and quantified by spectrophotometry and real-time PCR amplification of miR-21, miR-103, miR-24, and miR-125.

   Summary of Findings:

   Storage of CSF specimens at room temperature for 1 or 7 days had no discernable effect on EV yield, morphology, size distribution, or miRNA yield; or on levels of miR-21, miR-103, miR-24, and miR-125. In contrast, when EVs were lyophilized and stored at room temperature for 7 days there was an 80% reduction in EV count in two of three specimens evaluated. Despite the large decline in EV yields, no difference in EV size distribution or morphology were observed after room temperature storage of lyophilized EVs compared to EVs that were stored frozen. RNA yields were also comparable from lyophilized EVs stored at room temperature and EVs stored frozen, but the levels of the four individual miRNAs assessed were all lower in the lyophilized EVs. Although a single freeze-thaw cycle had no effect on EV count, morphology, size distribution, or recovery; RNA yield and levels of the four miRNAs quantified decreased with each subsequent freeze-thaw cycle.

    

   Biospecimens

   • Bodily Fluid - Cerebrospinal Fluid

   Preservative Types

   • Frozen
   • Other Preservative
   • None (Fresh)

   Diagnoses:

   • Neoplastic - Germ Cell

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   Morphology	Electron microscopy
   Cell count/volume	Light scattering
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Lyophilized Frozen
   Storage	Time at room temperature	1 day 3 days 7 days
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-21 miR-103 miR-24 miR-125
   Storage	Freeze/thaw cycling	0 cycles 1 cycle 2 cycles 3 cycles
   Storage	Storage temperature	-80°C Room temperature

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