Impact of Preanalytical and Analytical Methods on Cell-Free DNA Diagnostics.
Author(s): Krasic J, Abramovic I, Vrtaric A, Nikolac Gabaj N, Kralik-Oguic S, Katusic Bojanac A, Jezek D, Sincic N
Publication: Front Cell Dev Biol, 2021, Vol. 9, Page 686149
PubMed ID: 34552921 PubMed Review Paper? No
Purpose of Paper
This paper compared the yield, fragment size and methylation levels of cell-free DNA (cfDNA) isolated from pooled plasma and pooled seminal plasma using different methods with or without the addition of dithiothreitol (DTT). cfDNA was assessed by multiple methods including fluorometry, capillary electrophoresis, digital droplet PCR (ddPCR) and real-time PCR.
Conclusion of Paper
cfDNA yield and fragment size were dependent on specimen type (seminal plasma or plasma), extraction method (triton–heat–phenol (THP) method, several phenol–chloroform isoamyl alcohol (PCI) methods, salting-out, QIAmp Circulating Nucleic Acid Kit, Quick-cfDNA Serum & Plasma Kit, and NucleoSnap cfDNA Kit), and the method used for analysis. No single cfDNA isolation method proved to be superior in all assays. In general, inclusion of DTT was not beneficial during cfDNA extraction from plasma but did increase cfDNA yields from seminal plasma for some extraction methods. The level of methylation was not affected by cfDNA isolation method or the inclusion or exclusion of DTT, although seminal plasma displayed hypomethylation relative to plasma.
Studies
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Study Purpose
This study compared the yield, fragment size and methylation level of cfDNA isolated from plasma and seminal plasma using different methods with or without the addition of DTT. cfDNA was assessed by multiple methods including fluorometry, capillary electrophoresis, ddPCR, and real-time PCR. Blood was collected from twelve normozoospermic men into two EDTA tubes. Plasma was obtained by centrifugation of blood at 1400 x g for 10 min followed by 4500 x g for 10 min, within 2 h of blood collection. The same men collected ejaculate after five days of sexual abstinence, which was stored at room temperature for 45 min to 1 h to liquify. Seminal plasma was collected by centrifugation at 400 x g for 10 min, then 12,000 x g for 10 min, and then 20,000 x g for 10 min. Plasma and seminal plasma were pooled to create a single specimen of each type and stored at -80°C. cfDNA was extracted from plasma and seminal plasma using the triton–heat–phenol (THP) method, several published phenol–chloroform isoamyl alcohol isolation (PCI) methods, salting-out, a modified PCI method, QIAmp Circulating Nucleic Acid Kit, Quick-cfDNA Serum & Plasma Kit, and the NucleoSnap cfDNA Kit. cfDNA yield was quantified by Quant-iT PicoGreen dsDNA Assay Kit, capillary electrophoresis and ddPCR, and real-time PCR amplification of LINE-1. cfDNA size distribution was evaluated by real-time PCR amplification of long (224 bp) and short (82 bp) fragments of LINE-1 and by capillary electrophoresis with a cut-off off ≥224 bp. cfDNA was bisulfite converted with the EpiTect Plus DNA Bisulfite Kit and analyzed using the Pyromark Q24 Advanced System.
Summary of Findings:
On average, the commercial extraction kits yielded less plasma cfDNA than the THP, PCI, and salting out based methods (5-15 ng/mL versus 14-31 ng/mL). However, the commercial kits evaluated resulted in more copies than traditional isolation methods when quantified by capillary electrophoresis (15-27 ng cfDNA/mL versus 1-20 ng/mL), although the same was not true with fluorometric analysis. The highest cfDNA yield from plasma was obtained with the NucleoSnap cfDNA Kit. Similarly, real-time PCR and ddPCR amplified fewer copies of LINE-1 when extraction was with the THP, PCI, or salting out-based methods than when a commercial method was used (1-7 ng/mL versus 5-9 ng/mL and 1-6 ng/mL versus 4-7 ng/mL, respectively); the highest yields were obtained using QIAmp Circulating Nucleic Acid Kit and/or NucleoSnap cfDNA Kit. Addition of DTT during extraction with each of three commercial kits led to a small reduction in cfDNA yield (1-6%) and ddPCR amplifiable copies of LINE-1 (4-43%). The ratio of the 82 to 224 bp amplicon of LINE-1 varied considerably among the isolation methods evaluated but was highest when PCI methods were used for extraction. cfDNA fragment size by capillary electrophoresis was also highly variable, but the highest ratio of long to short fragments was observed with commercial kit extraction. There was no effect of cfDNA isolation method or addition of DTT on the level of methylation.
Overall, cfDNA yields from seminal plasma were similar among commercial extraction kits and THP, PCI, and salting out-based methods regardless of assay: fluormetric methods (86-656 ng/mL versus 95-727 ng/mL) or capillary electrophoresis (350-982 ng/mL versus 138-775 ng/mL). The highest cfDNA yields from seminal plasma were obtained by salting out, one of the PCI based methods, Quick-cfDNA Serum & Plasma Kit, and the NucleoSnap cfDNA Kit. cfDNA extraction from seminal plasma with a commercial kit resulted in more copies of LINE-1 by real-time PCR compared to THP, PCI, or salting out based methods (75–753 ng/ml versus 0.2-429 ng/mL), but effects on ddPCR amplifiable copies of LINE-1 were less clear (1-895 ng/mL using THP, PCI, and salting out based methods and 68-584 ng/mL using commercial kits). Addition of DTT during extraction with the QIAmp Circulating Nucleic Acid Kit and NucleoSnap cfDNA Kit led to higher cfDNA yields (14% and 33%, respectively) and more amplifiable copies by ddPCR (22% and 28%, respectively), but cfDNA yields and the amount of ddPCR amplifiable copies of LINE-1 were lower when DTT was added during extraction with the Quick-cfDNA Serum & Plasma Kit (-6% and -19%, respectively). cfDNA integrity, as assessed by the ratio of the 82 to 224 bp amplicon of LINE-1, was highly variable among the extraction methods examined but was highest when extraction was with one of the PCI methods or the Quick-cfDNA Serum & Plasma Kit without DTT. cfDNA fragment size by capillary electrophoresis was also highly variable, but the highest ratio of long to short fragments was observed when cfDNA extraction was with commercial methods and increased further with the addition of DTT. The level of methylation was not affected by cfDNA isolation method or the addition/exclusion of DTT, although seminal plasma was hypomethyled relative to plasma.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Capillary electrophoresis-MS DNA Bisulfite conversion assay DNA Digital PCR DNA Real-time qPCR DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Biospecimen location Seminal plasma
Plasma
Analyte Extraction and Purification Analyte isolation method Triton–heat–phenol (THP) method
Multiple phenol–chloroform isoamyl alcohol isolation methods (PCI)
Salting-out
Modified PCI method
QIAmp Circulating Nucleic Acid Kit
Quick-cfDNA Serum & Plasma Kit
NucleoSnap cfDNA Kit
DTT included
No DTT
Fluorometry Specific Technology platform Capillary electrophoresis
ddPCR
Real-time PCR
Real-time qPCR Specific Targeted nucleic acid LINE-1
Digital PCR Specific Technology platform 82 bp
224 bp
Digital PCR Specific Targeted nucleic acid LINE-1
Capillary electrophoresis-MS Specific Technology platform ddPCR