Verification of performance of a direct fluorescent assay for cell‑free DNA quantification, stability according to pre‑analytical storage conditions, and the effect of freeze‑thawing

Kim,Jae-Joon; Park,Kwonoh; Han,Yu Ran; Kim,Syed Hyun; Oh,Sang-Bo; Oh,So Yeon; Hong,Yun Jeong; Yun,Mi Sook

doi:10.3892/br.2021.1444

Verification of performance of a direct fluorescent assay for cell‑free DNA quantification, stability according to pre‑analytical storage conditions, and the effect of freeze‑thawing

Authors:
- Jae-Joon Kim
- Kwonoh Park
- Yu Ran Han
- Syed Hyun Kim
- Sang-Bo Oh
- So Yeon Oh
- Yun Jeong Hong
- Mi Sook Yun
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Affiliations: Medical Oncology and Hematology, Department of Internal Medicine, Pusan National University Yangsan Hospital, Yangsan‑si, Gyeongsangnam‑do 50612, Republic of Korea, Bionoxx Inc., Seongnam‑si, Gyeonggi, Gyeongsangnam‑do 50612, Republic of Korea, Department of Neurology, Uijeongbu St. Mary's Hospital, Catholic University of Korea, Seoul Special City, Gyeongsangnam‑do 50612, Republic of Korea, Division of Biostatistics, Research Institute for Convergence of Biomedical Science and Technology, Pusan National University Yangsan Hospital, Yangsan‑si, Gyeongsangnam‑do 50612, Republic of Korea
Published online on: June 15, 2021 https://doi.org/10.3892/br.2021.1444
Article Number: 68
Copyright: © Kim et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

A simple fluorescence‑based cell‑free DNA (CFD) assay has been previously developed that can directly measure nucleic acids without prior DNA extraction and amplification. However, studies on fluorescence‑based CFD are lacking. In particular, there is no known information regarding the stability with regard to pre‑analytical storage conditions in relation to time and temperature, or on the influence of freeze‑thawing. Plasma was directly assayed to measure CFD using PicoGreen™ reagent. Standard linearity and accuracy were confirmed using salmon sperm DNA. Whole blood was left at room temperature (RT) and at 4^˚C, and then plasma was separated. The CFD was also measured using thawed plasma after 1 week of freezing. As a correlation with a sperm DNA concentration, CFD demonstrated linearity over a wide range of concentrations, with a 0.998 correlation coefficient. The CFD level showed a change of up to 2.5 µg/ml according to pre‑analytical storage time, and the changes were not consistent over time. The CFD values at RT after 1 h were similar to the baseline values, and the relative standard deviation was lowest under this condition. The CFD values between 4˚C and RT were similar over all time periods assessed. After freeze‑thawing, the change in CFD value was reduced compared to that before freezing. The present study showed that CFD measurements using plasma processed within 1 h were optimal. Additionally, the effects of substantial changes according to storage conditions were reduced after freeze‑thawing, and thus studies using stored samples is viable and relevant.

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