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Comparison of Methods for MicroRNA Isolation from Extracellular Vesicles Obtained from Ascitic Fluids.

Author(s): Skryabin GO, Vinokurova SV, Elkina NV, Denisova DA, Beliaeva AA, Zhordania KI, Bagrov DV, Enikeev AD, Galetsky SA, Komelkov AV, Krasnoshekova GI, Tchevkina EM

Publication: Biochemistry (Mosc), 2022, Vol. 87, Page 1354-1366

PubMed ID: 36509726 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare the yield and percentage of miRNA, small RNA concentration, next generation sequencing results and levels of individual miRNA among RNA extracts obtained using six different isolation methods from ascites fluid extracellular vesicles (EVs). Within 1 h of collection, ascites fluid from six patients with ovarian cancer was sequentially centrifuged at 4°C for 15 min at 300 g, 20 min at 800 g, 30 min at 2000 g and 30 min at 10,000 g to remove cells and debris. The supernatant was then stored at -80°C until small EV isolation by ultracentrifugation (110,000 g for 3 h followed by recentrifugation of aliquots of the resuspended pellet at 110,000 g for 1 h). The EV pellet was then resuspended in PBS or PureLink Binding buffer (for PureLink RNA extraction) and EVs were quantified by nanoparticle tracking analysis and morphology was evaluated by transmission electron microscopy. The protein expression of isolated EVs was investigated by Western Blotting using antibodies against Flotillin-2, CD9, TSG-101 and PCNA.  miRNAs were extracted from EVs using the Total Exosome RNA & Protein Isolation Kit with both one and two columns, miRNeasy Serum/ Plasma Kit, PureLink miRNA Isolation Kit, and the miRNeasy Advanced Serum/Plasma Kit. Small RNAs were quantified using the Qubit microRNA Assay Kit and size distribution was evaluated by bioanalyzer. Sequencing libraries were constructed using NEBNext Multiplex Small RNA Library Prep Set and single-end sequenced on a HiSeq1500 instrument. Levels of miR-1246, miR-200b, miR-23a, and miR-200c were quantified by real-time RT-PCR.

   Summary of Findings:

   The mean size of the isolated EVs varied from 118-158 nm. The ascites EVs had the proper deflated ball morphology, had high levels of the three exosomal proteins investigated (CD9, Flotillin-2 and TSG-101) and lacked detectable PCNA (the cell-derived marker).  When the PureLink miRNA Isolation Kit (PBS and binding buffer EVs), miRNeasy Serum/Plasma Kit and the Total Exosome RNA & Protein Isolation Kit with column 1 or 2 were compared, the highest miRNA concentration (Bioanalyzer) was achieved using the Total Exosome RNA & Protein Isolation Kit with column 1 followed by the Total Exosome RNA & Protein Isolation Kit with column 2, while the lowest yield was achieved using the PureLink Kit regardless of whether the EVs were in PBS or Binding buffer. Although, the yield from all three specimens was significantly lower using column 2 rather than column 1 when determined by bioanalyzer (P<0.05), when analyzed using the Qubit assay yields were comparable using column 1 and column 2 with the total Exosome RNA & Protein Isolation Kit.  Although there were no significant differences in the percentage miRNAs among the kits (PureLink-PBS, miRNeasy, and Total Exosome RNA & Protein Isolation Kit with column 1), the percentage was highest when the Total Exosome RNA & Protein Isolation Kit with column 2 was used for all three specimens evaluated. When the miRNeasy Serum/Plasma Kit, miRNeasy Advanced Serum/Plasma Kit, and Total Exosome RNA & Protein Isolation Kit with column 1 were compared, the miRNeasy Advanced Kit yielded the most miRNA, although the percentage of miRNA and the concentration of small RNA were comparable among the three kits. Further, the Cq values for individual miRNAs were comparable or higher when extraction was with miRNeasy Advanced rather than Total Exosome RNA & Protein Isolation Kit with column 1.  The majority of reads mapped to lncRNAs (31-39%) or mRNAs (22-32%), regardless of extraction method. The rRNAs (5-19%), miRNAs (1-10%), and miscellaneous RNAs percentages were variable among the kits examined, with the highest percentage of reads that mapped to miRNA achieved using the Total Exosome RNA & Protein Isolation Kit with column 1. While 68.4% of miRNA detected were quantified in RNA extracted from all kits (Total Exosome RNA & Protein Isolation Kit with column 1, miRNeasy and PureLink), an additional 7.7%, 7.7%, and 10.3% of miRNA were only detected in extracts from the miRNeasy, PureLink and Total Exosome RNA & Protein Isolation Kit with column 1, respectively.

    

   Biospecimens

   • Bodily Fluid - Ascites

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Next generation sequencing
   RNA	Fluorometry
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-1246 miR-200b miR-23a miR-200c
   Analyte Extraction and Purification	Analyte isolation method	Total Exosome RNA & Protein Isolation Kit with column 1 Total Exosome RNA & Protein Isolation Kit with column 2 miRNeasy Serum/Plasma Kit miRNeasy Advanced PureLink from EVs in binding buffer PureLink from EVs in PBS

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