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A novel assay for exosomal and cell-free miRNA isolation and quantification.

Author(s): Grunt M, Failla AV, Stevic I, Hillebrand T, Schwarzenbach H

Publication: RNA Biol, 2020, Vol. 17, Page 425-440

PubMed ID: 31986967 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to identify the optimal conditions for MGP-based exosome enrichment from plasma, serum, and urine and exosomes isolated using this method were compared to those obtained using the Total Exosome Isolation Reagent Kit. Blood was collected from an unspecified number of volunteers into S-Monovette Z (Serum) and S-Monovette K₃EDTA tubes and centrifuged at 300 x g for 10 min followed by recentrifugation of the plasma and serum at 2,000 x g for 10 min and then 10,000 x g for 10 min. The plasma and serum were filtered through Whatman Puradisc 25 syringe filters. The authors report the use of urine but details were not provided. Optimization of exosome enrichment involved optimization of the first reagent (MGP 1, 2, 3, 4, or 5) and its volume (20, 30, 40, 50, 60, 90, or 100 µL), incubation duration (0, 1, 10, 30, or 60 min), temperature (4°C, room temperature, or 50°C), and condition (no shaking or 400 rpm); the second reagent (calcium chloride, calcium acetate, ammonium chloride, zinc chloride, or manganese chloride) and its volume (100, 150, 300, or 450 µL), incubation time (3, 10, 30, or 60 min), temperature (4°C, room temperature, or 50°C) and condition (no shaking or 400 rpm); the centrifugation protocol (1 min at 8,000, 10,000 or 16,000 x g; 2 min at 5000, 10,000 or 16,000 x g; 3 min at 16,000 x g; 10 min at 500 x g; 15 min at 5,000 or 16,000 x g; 30 min at 3,000, 5,000, 10,000, or 16,000 x g; or 60 min at 16,000 x g), the post-centrifugation steps (washing of the pellet, a second 10 sec spin, or no additional steps) and the resuspension fluid [ddH2O, PBS,  RIPA, tri-natriumcitrat (50 mM, 100 mM, 150 mM, or 200 mM), EDTA (50 mM, 100 mM, or 1 M), or PBS with 50 mM EDTA]. Exosomes were also isolated from plasma and serum with Total Exosome Isolation Reagent Kit.  Protein concentrations were measured by Bradford assay and the presence of CD63 confirmed by Western blotting. Exosome size was confirmed by nanoparticle tracking analysis (NTA) and visualized by confocal microscopy after staining with Exo-Glow Exosome Labelling Kit. miRNA was extracted using an in-house optimized method (see study 2) and levels of miR-16 and miR-142 levels quantified by an in-house developed real-time RT-PCR assay.

   Summary of Findings:

   The lowest quantification cycle (Cq) values were obtained when exosomes were extracted from 500 µL plasma with 30 µL room temperature MGP1 followed by incubation with 150 µL 1 M calcium chloride at room temperature and pelleting of the exosomes by centrifugation at 16000 x g for 30 min followed by 16000 x g for 10 sec and dissolving the pellet in 50 mM EDTA. The authors report similar findings for serum as plasma. Importantly, Western blot confirmed the presence of clear bands for the exosome marker CD63 following isolation of exosome from plasma and serum but not urine using this method. The size of the isolated exosomes was 20-300 nm consistent with exosomes and exosome aggregates as larger microvesicles were removed prior to the addition of MGP by filtration through a 0.22 µm filter. The MGP-based protocol was also able to isolate exosomes from SEC fractions, indicating it can be used for highly dilute samples without co-precipitation. Importantly, the authors state that exosomes purified using the optimized MGP-based method were not as yellow as those obtained using the commercial kit, indicating less contamination with plasma proteins. The protein levels were 5.1 and 2.3-fold higher in plasma and serum, respectively, when isolated using the commercial kits rather than the optimized MGP-based method. Real-time RT-PCR found significantly higher levels of miR-16 in plasma supernatant than exosomes when extraction was with the MGP-based method (P=00001), but serum miR-16 levels were higher in supernatant relative to than exosome using either the MGI-based method (P=0.004) and the commercial kit (P=0.009).  miR142 levels were higher in exosomes than in the supernatant of plasma and serum when either the optimized MGP-based method (P=0.0001, both) or commercial kit were used (P=0.028 and P=0.0001, respectively).

   Biospecimens

   • Bodily Fluid - Urine
   • Bodily Fluid - Blood
   • Bodily Fluid - Serum
   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   Protein	Western blot
   Protein	Colorimetric assay
   Cell count/volume	Light microscopy
   Cell count/volume	Light scattering

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Biospecimen location	Plasma Serum Urine
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Plasma exosomes Serum Serum exosomes
   Western blot Specific	Targeted peptide/protein	CD63
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-16 miR-142
   Analyte Extraction and Purification	Analyte isolation method	MPG exosome enrichment Total Exosome Enrichment Kit Exosome enrichment optimization using MPG

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2. Study Purpose

   The purpose of this study was to optimize the miRNA isolation method from exosomes, plasma, and serum. Blood was collected from an unspecified number of volunteers into S-Monovette Z (Serum) and S-Monovette K₃EDTA tubes and centrifuged at 300 x g for 10 min followed by recentrifugation of the plasma and serum at 2,000 x g for 10 min and then 10,000 x g for 10 min. The plasma and serum were filtered through Whatman Puradisc 25 syringe filters. Exosomes were purified from plasma and serum using the optimized MGP-based method (Study 1). Plasma, exosomes, or serum were lysed in different volumes (200, 300, 400, 500, or 600 µL) of different buffers (from the PME free-circulating DNA Extraction Kit, the innuCONVERT Bisulfite Body Fluids Kit,  Analytik Jena, innuPREP Plant DNA Kit innuPREP FFPE DNA Kit, innuPREP Blood DNA MIDI Direct Kit, innuCONVERT Bisulfite All-in-One Kit, Instant Virus RNA/DNA Kit, innuPREP Blood DNA Mini Kit, innuPREP Blood DNA Midi Kit, innuPREP RNA mini Kit, innuPREP Stool DNA Kit, innuSPEED Soil DNA Kit, innuPREP Plant DNA/RNA Virus Kit, innuPREP Plant RNA Kit, innuPREP FFPE total RNA Kit, or innuPREP Virus DNA/RNA Kit) or in a variety of homemade lysis solutions consisting of different combinations of SDS (1%, 5%, 10%), Tris-HCl pH 8.0 (1.3 M, 2.6 M, or 50 mM) , urea (0.3 M, 0.6 M, or 95 mM), EDTA (1 mM, 10mM, 25 mM, 50 mM, or 100 mM), or Na-citrate (5 mM, 10 mM, 15 mM, 20 mM, or 50 mM), CaCl2 (1 mM) , Tween-20 (0.5%), GSCN (4 or 5%) supplemented with proteinase K (10, 20, 30, 40, or 50 µL) at different temperatures (room temperature for 5 min; 70°C for 10, 15, 20, or 30 min; 55° C for 20 min; or 85°C for 20 min). Specimens were then centrifuged at 16,000 x g for different durations (1, 2, 3, 5, or 10 min) and the supernatant mixed with binding buffers (400, 600 or 800 µL ethanol; 400, 600, or 800 µL isopropanol; 400 or 600 µL 55% tetrahydrofuran; 600 µL 100% tetrahydrofuran; 400 µL RBF from the innuPREP DNA Sizing Kit; 200 μl VL from Analytik Jena; or 400 µL SBS from the innuPrep Plant DNAKkit) before application to various spin columns from Analytik Jena. The columns were washed with different wash solutions from the blackPREP FFPE DNA Kit, innuPREP DNA Mini Kit, innuPREP Blood RNA Kit, innuCONVERT Bisulfite Body Fluids Kit, or 650 μl LS from the innuPREP Blood RNA Kit alone or in combination with or without additional ethanol wash, centrifuged at 16,000 x g for 3 min and eluted using different amounts (50 or 100 μl) of various elution buffers (innuPREP Blood DNA Mini Kit or water) for different durations (1, 2, 10, or 20 min) at different temperatures (room temperature, 40°C, or 70°C) followed by re-application of the flow-through once or twice. miRNA yield was confirmed by real-time PCR amplification of miR-16 and miR-142 using Applied Biosystem and an in-house assay with modified reverse transcription (9.375 units RT-enzyme, 250 nm stem-loop primer, addition of DTT to buffer, exclusion of RNAse inhibitor, and inactivation at 95°C) and real-time RT-PCR (SpeedAmp Optimization Buffer, optimized primer and probe concentrations, optimized thermocycler program).

   Summary of Findings:

   The lowest Cq values were found when RNA was isolated from exosomes lysed for 20 min at 55°C in 400 μl of a lysis buffer containing 5% SDS, 1.3 M Tris-HCl pH 8.0, 0.3M urea, 5 mM Na-citrate, supplemented with 1 mM CaCl2, and 20 μl proteinase K. The CCq values were further optimized by centrifugation of the mixture at 16,000 x g for 5 min and mixing the supernatant with 800 μl absolute ethanol and loaded twice on the spin column. The optimal spin column protocol included centrifugation at 11,000 x g for 1 min after loading of the solution and washing of the column with 500 μl HS from the innuPREP DNA Mini Kit and 650 μl LS from the innuPREP Blood RNA Kit and centrifugation. After washing of the column, Cq values were lowest when eluted in 100 μl of RNase-free water for 10 min at room temperature and re-elution of the flow-through for an additional 10 min at room temperature. The authors report similar results for serum and plasma. After isolation using this protocol, use without dilution led to over-amplification using the Applied Biosysems real-time RT-PCR protocol (efficiency 119.1%) but the efficiency of amplification using the in-house method was 99.7%.

   Biospecimens

   • Bodily Fluid - Serum
   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Serum Serum exosomes Plasma exosomes
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-16 miR-142
   Real-time qRT-PCR Specific	Technology platform	In-house optimize real-time RT-PCR method Applied Biosystems real-time RT-PCR protocol
   Analyte Extraction and Purification	Analyte isolation method	Isolation optimization performed

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