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Stability of Circulating Exosomal miRNAs in Healthy Subjects.

Author(s): Sanz-Rubio D, Martin-Burriel I, Gil A, Cubero P, Forner M, Khalyfa A, Marin JM

Publication: Sci Rep, 2018, Vol. 8, Page 10306

PubMed ID: 29985466 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of frozen storage before or after exosomal isolation and exosome isolation kit on exosome size and miRNA expression. EDTA blood was collected using a 21-gauge needle from 7 healthy subjects over the course of four weekly visits. Plasma was obtained by centrifugation at 3000 x g at 4˚C for 15 min. Plasma was then frozen at -80˚C or used fresh for exosome isolation. Exosomes were isolated using the miRCURY Exosome Isolation kit and Total Exosome Isolation kit. Isolation of exosomes was confirmed using dynamic light scattering, transmission electron microscopy (TEM) and by Western blot analysis (CD63 and HSP70 positive and GM130 negative). Exosomes obtained from fresh plasma were stored at -20˚C, while RNA was extracted directly from those from frozen plasma. RNA was isolated using the miRCURY RNA Exosome Isolation Kit and quantified by real-time PCR.

   Summary of Findings:

   The majority of the exosomes were 30-50 nm with an additional group of exosomes that were 130-150 nm, regardless of extraction method. However, exosomes that were frozen after extraction from fresh plasma had larger particles (up to 300-480 nm, depending on extraction method). TEM confirmed the presence of small spheres between 30 and 150 nm in all specimens, but when the exosomes were frozen instead of the plasma there was a lot of background and the authors report more salt precipitates.

   Using the BestKeeper, Normfinder and geNorm software the authors identified let-7a to be the most stably expressed exosomal miRNA. Generally, miRNA CT values were higher when exosomes were frozen rather than plasma with significantly higher CTs observed for let-7a, miR-16, miR-126, and miR-320 regardless of extraction kit, miR-145 and miR-222 when extracted using miRCURY Exosome Isolation kit and miR-21 and miR-150 when extracted using the Total Exosome Isolation kit.  Further miR-143 and miR-145 were not quantifiable when exosomes isolated with the Total Exosome Isolation kit were frozen. The inter-individual coefficient of variance was lower in exosomes extracted using miRCURY Exosome Isolation kit than the Total Exosome Isolation kit, but level of variance was miRNA specific. Further, while levels of many miRNA differed significantly between visits when extracted using the Total Exosome Isolation kit, only miRNA-145 differed significantly when extracted using the miRCURY Exosome Isolation kit, regardless of whether plasma or exosomes were frozen. Bland-Altman analysis showed the variability was highest when exosomes were extracted from frozen plasma using the Total Exosome Isolation kit, and that there was concordance of 98% for all miRNA, except miR-14,5 when extracted using miRCURY Exosome Isolation kit. The percentage difference between miRNA from frozen exosomes and frozen plasma were within the 95% confidence interval for 90% of the miRNA. The average CVs for the miRNA between the four visits was lowest when extraction was from frozen plasma with the miRCURY Exosome Isolation kit.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Normal

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Total Exosome Isolation Kit miRCURY Exosome Isolation Kit
   Storage	Storage conditions	Frozen as plasma Frozen as exosomes
   Biospecimen Preservation	Type of fixation/preservation	Frozen None (fresh)

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