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

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

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.

Keywords: biomarkers; cell-free nucleic acids; fluorescence; freezing; polymerase chain reaction.

Conflict of interest statement

The authors declare that they have no competing interests.

Copyright: © Kim et al.

Figures

Figure 1
Figure 1
Correlation between the direct fluorescent assay and PCR method for measurement of CFD. As a result of analyzing the average fluorescence intensity correlation with the sperm DNA concentration, quantification of plasma CFD demonstrated linearity over a wide range of concentrations (1-1,000 ng/ml) with a correlating standard curve (R2=0.998). RSD, relative standard deviation; CFD, cell free DNA.
Figure 2
Figure 2
Fluorescence-based CFD according to varying storage condition. At both RT and 4˚C, the CFD values increased after 1 h, decreased at 2 h, increased after 3 h and then decreased after 6 h. The changes in CFD values were significant according to time course. F=481.038, P

Figure 3

Influence of freeze-thawing in the…

Figure 3

Influence of freeze-thawing in the fluorescence-based CFD assay. Plasma samples were stored frozen…

Figure 3
Influence of freeze-thawing in the fluorescence-based CFD assay. Plasma samples were stored frozen as aliquots at -80˚C and thawed after 1 month to assess the influence of freezing, and the reproducibility of the results. There was a significant difference in CFD values after freeze-thawing according to storage time. F=3.387, P=0.007. However, there was no significant difference in CFD values after freeze-thawing based on the previously exposed temperature. F=1.342, P=0.280. RSD, relative standard deviation; CFD, cell free DNA; RT, room temperature.
Figure 3
Figure 3
Influence of freeze-thawing in the fluorescence-based CFD assay. Plasma samples were stored frozen as aliquots at -80˚C and thawed after 1 month to assess the influence of freezing, and the reproducibility of the results. There was a significant difference in CFD values after freeze-thawing according to storage time. F=3.387, P=0.007. However, there was no significant difference in CFD values after freeze-thawing based on the previously exposed temperature. F=1.342, P=0.280. RSD, relative standard deviation; CFD, cell free DNA; RT, room temperature.

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