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.

Keywords: CSF; EV; exosome; freeze thaw; lyophilization; stability.

Fig. 1. Clinical CSF EVs stored under Room Temperature (RT) and −80°C

EVs isolated from CSF that were stored at room temperature for 1 and 7 days were comparable to EVs isolated from CSF stored at −80°C. (A) Total number of EVs recovered as determined by Nanoparticle tracking analysis (NTA). (B) Representative TEM images of EVs isolated from CSF stored under different conditions. Scale bar represents 500nm in 5000× magnification images, and 200nm in 25000× magnification images. (C) EV size profile as determined by NTA. (D) Comparison of total EV RNA yield extracted using Qiagen miRNeasy mini kit. (E) Detection of miR-21, miR-24, miR-103 and miR-125 transcripts in EVs by qRT-PCR. EV miRNAs appeared to be stable in CSF at room temperature for up to 7 days. Experiments were performed in triplicate and data are expressed as mean ± SEM.

Fig. 2. Effects of lyophilization on clinical CSF EV stability and EV associated miRNAs

Lyophilized CSF EV samples were stored at room temperature for 7 days prior to rehydration with distilled water and then subjected to parallel analysis against non-lyophilized EVs. (A) Total number of EVs recovered as determined by NTA. (B) Representative TEM images of non-lyophilized EVs and EVs that were lyophilized and rehydrated. Scale bar represents 500nm in 5000× magnification images, and 200nm in 25000× magnification images. (C) EV size profile as determined by NTA. (D) Comparison of total EV RNA yield extracted using Qiagen miRNeasy mini kit. (E) Detection of miR-21, miR-24, miR-103 and miR-125 transcripts in EVs by qRT-PCR. Significant reductions in miRNA levels were observed in lyophilized EVs. Lyophilization was performed in duplicate and data are expressed as mean ± SEM. * indicates p<0.05 as determined by Student’s t-test.

Fig. 3. Effects of multiple freeze-thaw cycles on clinical CSF EV

CSFs were subjected to multiple rounds of freezing and thawing prior to EV isolation. Recovered EVs were characterized by (A–B) NTA, and (C) TEM. Scale bar represents 500nm. Inset shows a magnified view of the EV. (D) EV RNA was then extracted and (E) miRNA levels were quantitated by qRT-PCR. 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. Experiments were performed in triplicate and data are expressed as mean ± SEM. * indicates p<0.05 as determined by Student’s t-test.