Quantification of Cell-Free DNA: A Comparative Study of Three Different Methods

Jeon, Kibum; Lee, Jiwon; Lee, Jee Soo; Kim, Miyoung; Kim, Han Sung; Kang, Hee Jung; Lee, Young Kyung

Abstract

BACKGROUND
Cell-free DNA (cfDNA) provides unique potential as a biomarker for cancer patients or in the field of prenatal care. Following extraction from plasma, cfDNA should be quantified before downstream analysis. The standardization of the quantification methods is essential for application of these techniques to laboratory practice. We aimed to compare three different quantification methods (spectrophotometry, fluorometry, and the electrophoresis-based method) and to determine the contributor(s) to the differences, if any.
METHODS
A total of 135 plasma samples obtained from cancer patients (n=71) and normal individuals (n=15) and cfDNA were extracted from the samples. The extracted cfDNA was quantified using three different methods: NanoDrop spectrophotometry, Quantus fluorometry, and the electrophoresis-based 4200 TapeStation method.
RESULTS
NanoDrop exhibited the highest estimates (median, 12.3 ng/uL). The estimates of Quantus (median, 0.33 ng/uL) were higher than those of 4200 TapeStation (median, 0.22 ng/uL). Quantus and TapeStation were significantly correlated (r=0.802, P<0.001). The Bland-Altman plot showed a positive bias of 0.24 ng/uL for Quantus compared with TapeStation. The difference between Quantus and TapeStation values demonstrated a significantly moderate correlation with the amount of high molecular weight DNA (r=0.357, P<0.001).
CONCLUSIONS
Nanodrop is considered as a non-specific method of cfDNA quantification. Quantus depicted more specific quantification compared to Nanodrop; however, TapeStation may be used in parallel or as a complementary method as it enables to determine the size distribution of DNA fragments.