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Centrifugation-free extraction of circulating nucleic acids using immiscible liquid under vacuum pressure.

Author(s): Lee H, Na W, Park C, Park KH, Shin S

Publication: Sci Rep, 2018, Vol. 8, Page 5467

PubMed ID: 29615736 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the cfDNA extraction efficiency of the QIAamp circulating nucleic acid kit and the author’s PIBEX method and compared the size of the cfDNA peaks generated from bioanalyzer results. Plasma was isolated from the K₂EDTA blood of seven healthy patients by centrifugation at 1,900 x g for 10 min followed by recentrifugation of the supernatant at 12,000 x g for 15 min. Proteinase K and lysis buffer were added to the plasma and the mixture was incubated at 60°C for 30 min. Binding buffer was then added and the mixture was placed on ice for 5 min. The cfDNA was then extracted using the remaining steps in the QIAamp circulating nucleic acids kit using a QIAvac 24 plus and using the author’s PIBEXsystem. The PIBEX method used mineral buffer above the elution buffer layer and used a switch to apply the wash buffers, ethanol, and elution buffer all through a low-pressure vacuum. A drying step was included in which room temperature air was pulled through the vacuum for 3 min. cfDNA was quantified by real-time PCR and DNA purity and size were confirmed by bioanalyzer.

   Summary of Findings:

   The concentration of each of the four cfDNAs was comparable in specimens extracted using the author’s PIBEX system and the QIAamp kit for each of the seven samples. There was a trend toward higher concentrations of GAPDH, TERT, and NAGK when extracted with PIBEX but differences were not significant. Further, the bioanalyzer traces were comparable between methods with a similar location of the cfDNA peak (172 bp for QIAamp and 169 for PIBEX).

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR
   DNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Real-time qPCR Specific	Targeted nucleic acid	GAPDH TERT RPPH1 NAGK
   Analyte Extraction and Purification	Analyte isolation method	QIAamp circulating nucleic acid kit PIBEX system

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