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An automated, high throughput methodology optimized for quantitative cell-free mitochondrial and nuclear DNA isolation from plasma.

Author(s): Ware SA, Desai N, Lopez M, Leach D, Zhang Y, Giordano L, Nouraie M, Picard M, Kaufman BA

Publication: J Biol Chem, 2020, Vol. , Page

PubMed ID: 32900851 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study identified the optimal plasma volume, proteinase K and SDS concentrations, and digestion temperature and duration for isolation of mtDNA and nDNA from a single pooled plasma specimen. Venous EDTA blood from an unspecified number of patients (no diagnosis provided) was centrifuged at 950 x g for 15 min and plasma was pooled, aliquoted, and then stored at -80˚C. Unless otherwise specified, 300 µL of a single plasma pool was digested for 16 h at 70˚C with 750 ng/µL proteinase K in 0.93% SDS and extraction was performed using the MagMAX Cell Free DNA Kit with a DynaMag-2. All experiments were performed in triplicate. To test the requirement for proteinase K, mtDNA and nDNA were extracted directly from plasma with and without a 22 min proteinase K digestion. To identify the optimal duration of proteinase K digestion, plasma pools were incubated in SDS with proteinase K for 22 min, 1 h, or 16 h at 70˚C. To identify the optimal proteinase K concentration, digestion was performed on 75 µL of plasma using 750, 1500, and 3000 ng/µL proteinase K. To identify the ideal plasma volume, DNA was extracted from 37.5, 75, 150, and 300 µL plasma. To identify the ideal concentration of SDS, 75 µL plasma was subjected to proteinase K digestion in 0, 0.47%, 0.93%, and 1.86% SDS. To identify the ideal digestion temperature, 75 µL of plasma was digested at 55˚C and 70˚C. mtDNA and nDNA were quantified by TaqMan duplex real-time PCR assays and mitochondrial/nuclear primer pairs for NADH:ubiquinone oxidoreductase core subunit 1(ND1)/beta-2-microglobulin (B2M) and NADH core subunit 4 (ND4)/nuclear-encoded human peptidylprolyl isomerase A (PPIA).

   Summary of Findings:

   Inclusion of a proteinase K step significantly increased yield of both mtDNA and nDNA (P<0.05 and P<0.01, respectively).  Similarly, extraction for 16 h rather than 22 min resulted in significantly higher yields of mtDNA and nDNA (P<0.05 and P<0.01, respectively) and the yield of nDNA was higher following a 1 h digestion than a 22 min digestion (P<0.05) but only a non-significant increase when digestion was extended from 1 to 16 h. Extraction at 70˚C rather than 55˚C significantly increased the yield of mtDNA and nDNA (P<0.01 and P<0.05, respectively). Use of ≤150 µL of plasma resulted in 8-fold more mtDNA (P<0.001) and a non-significant 4-fold increase in nDNA. Elimination of SDS rather than inclusion at any other tested concentration resulted in reduced yield (P<0.001, all). There was no effect of proteinase K concentration on the yield of either mtDNA or nDNA.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Protein digestion	In 0.47% SDS In 0.93% SDS In 1.86% SDS No Proteinase K 1500 ng/µL proteinase K 750 ng/µL proteinase K 3000 ng/µL proteinase K At 55˚C At 70˚C 22 min digestion 1 h digestion 16 h digestion
   Biospecimen Aliquots and Components	Aliquot size/volume	300 µL 150 µL 75 µL 37.5 µL

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2. Study Purpose

   This study identified the optimal bead volume, lysis solution volume, elution time and volume, bead incubation time, mixing speed, and inclusion of isopropanol for isolation of mtDNA and nDNA from a single pooled plasma specimen. Venous EDTA blood from an unspecified number of patients (no diagnosis provided) was centrifuged at 950 x g for 15 min and plasma was pooled, aliquoted, and then stored at -80˚C. A 75 µL aliquot of plasma was digested with 1500 ng/µL Proteinase K for 16 h at 70˚C in 1.86% SDS. Unless otherwise specified, extraction was performed using 125 µL lysis binding solution and 3 µL magnetic beads using the MagMAX Cell Free DNA Kit with a DynaMag-2. To identify the effect of altering the volume of the reagents based on the 75 µL plasma volume, extraction was performed using 125 µL lysis binding solution, 3 µL magnetic beads, 265 µL wash 1, 475 µL wash 2 instead of 405 µL lysis binding solution or 10 µL magnetic beads, 450 µL wash 1, and 900 µL wash 2. To investigate the effects of magnetic bead volume, 2.5, 5, 10, and 15 µL magnetic beads were added to 250 µL lysis binding solution. To investigate the effects of lysis binding solution volume, extraction was conducted using 101.25, 250, and 643 µL lysis buffer. To investigate the effects of an additional bead step, a second incubation was performed for 0 or 10 min with and without shaking. To investigate the effects of elution volume and time, elution was conducted using 60 µL and 125 µL elution solution and elution was performed for 6 or 20 min. The effects of mixing fast versus slow and the inclusion of 50% isopropanol in the lysis solution were also investigated. mtDNA and nDNA were quantified by TaqMan duplex real-time PCR assays and mitochondrial/nuclear primer pairs for ND1/ B2M and ND4/ PPIA.

   Summary of Findings:

   The optimized lysis binding solution, magnetic bead, and wash volumes resulted in higher yields of nDNA but had no effect on mtDNA yields. However, yields were not affected by lysis binding solution or magnetic bead volume when adjusted separately. Similarly, there was no effect of an additional bead step including 10 min of incubation, regardless of mixing. Use of a faster mixing speed rather than the slower speed increased the yield of mtDNA but did not affect nDNA yield. Inclusion of isopropanol in the lysis solution decreased the yield of mtDNA and nDNA. Doubling the elution volume had no effect on the yield beyond the expected 1 CT increase (2-fold change reflecting volume difference). Similarly, increasing the elution time from 6 to 20 min did not affect the yield.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Extraction using original volumes Extraction using optimized volumes based on plasma volume 2.5 µL magnetic beads 5 µL magnetic beads 10 µL magnetic beads 15 µL magnetic beads 101.25 µL lysis binding buffer 250 µL lysis binding buffer 643 µL lysis binding buffer No additional beads Extra 10 min with beads no shaking Extra 10 min with beads with shaking 60µL elution solution 120µL elution solution Elution 6 min Elution 20 min MPP mixing fast MPP mixing slow Isopropanol added to buffers No isopropanol added to buffers

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3. Study Purpose

   The purpose of this study was to investigate the sources of variation in nDNA and mtDNA yield from plasma. Venous EDTA blood from an unspecified number of patients (no diagnosis provided) was centrifuged at 950 x g for 15 min and plasma was pooled, aliquoted, and then stored at -80˚C. A 75 µL aliquot of plasma was digested with 1500 ng/µL proteinase K for 16 h at 70˚C in 1.86% SDS. Extraction was performed using 125 µL lysis binding solution and 3 µL magnetic beads using the MagMAX Cell Free DNA Kit with a magnetic particle processor (MMP) and liquid handler. To identify the extent of variability, cfDNA was extracted from the same plasma specimen daily for 5 days and then in 96 replicates on one plate. To investigate the source of edge effects, plasma was digested prior to loading on the 96 well plate; processing was conducted at room temperature, 24˚C, and 30˚C; and use of an alternate protocol involving a single bead step with 1 min fast mixing and 8 min medium mixing instead of two mix steps for 6 min fast. To investigate if variability within the plate may be due to viscous fluids being dispensed improperly by the liquid handler, SDS and lysis binding solution were manually pipetted instead of using the liquid handler. mtDNA and nDNA were quantified by TaqMan duplex real-time PCR assays and primer pairs for ND4 and B2M and ND4 and PPIA.

   Summary of Findings:

   The variability between replicate extractions performed over 5 days was higher than expected. When cfDNA was isolated from the same plasma specimen 96 times on one plate, the standard deviation (Std Dev) in nDNA and yield was 0.413 and the Std Dev in mtDNA yield was 0.927. Further investigation showed that less mtDNA was obtained from the edges and corners of the plate than the middle but nDNA yield was uniform. No effect of plate position was observed on PCR amplification confirming the effect arose during extraction and was not due to liquid handler. Digestion of the plasma prior to extraction showed that a third of the variability in mtDNA yield was due to the digestion step (Std Dev 0.667 for mtDNA and 0.30 nDNA when predigested), but digestion before extraction did not resolve the edge effects. Heating the plate to 24˚C for all steps rather than at room temperature or 30˚C, resolved some of the edge effects and decreased the Std Dev to 0.387 for mtDNA and 0.256 for nDNA, but an effect was still noted for one corner. Variability between replicates was no longer observable after switching to a different MPP protocol that included a change in mix time (1 min fast and 8 min medium instead of 6 min fast), only one bead step instead of two, and the Std Dev was reduced to 0.163 for mtDNA and 0.172 for nDNA. Manual pipetting of SDS and Proteinase K rather than use of the liquid handler improved mtDNA recovery (P<0.0001).

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Location on plate during extraction Plate at room temperature Plate at 24˚C Plate at 30˚C Alternate extraction protocol with 1 bead step Original extraction protocol with 2 bead steps
   Analyte Extraction and Purification	Protein digestion	On plate Prior to placing on plate Manual pipetting of SDS and Proteinase K Liquid handler for SDS and Proteinase K
   Real-time qPCR Specific	Specimen position	Center of plate Periphery of plate

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