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

Abstract

Cell-free DNA (cfDNA) is a subset of circulating extracellular DNA in plasma, which is released from cells mostly through apoptosis, necrosis, and active secretion [1]. The cfDNA is easily obtained by using minimally invasive procedures and has unique potential biomarkers for non-invasive prenatal testing and cancer management [2,3]. The recent advances in molecular technologies such as next-generation sequencing (NGS) and digital polymerase chain reaction (dPCR) have enabled routine analysis in clinical laboratories [4]. The standardization of the analytical conditions to detect the genetic alterations in cfDNA still remains a major challenge [5]. After isolation from plasma, cfDNA is quantified before downstream analysis (e.g., NGS and dPCR). An accurate measurement of cfDNA, as a stringent quality control of cfDNA isolation, is a critical preliminary step for these downstream analyses [6]. Several methods have been described to quantify cfDNA. Quantitative PCR (qPCR) has been used to measure DNA concentration by targeting various pre-selected DNA sequences, and recently more rapid and costeffective methods such as spectrophotometry, fluorometry, and electrophoresis-based methods are being used [5].

Technical and Methodological Aspects of Cell-Free Nucleic Acids Analyzes

“…Measurement of absorbed or transmitted light can be used to measure the amount of a known chemical substance considering each chemical compound absorbs or transmits light over a particular wavelength range, while not only NAs have specific absorbance ranges, but also possible contaminants of the sample, such as chemicals or proteins. This can be considered for a drawback, since different substances may have the same or similar absorbance spectra, possibly leading to signal overlap and concentration overestimation [ 268 ], but also for an advantage, with an ability to provide purity measurements of the analyzed samples. Contaminants may represent spectrophotometry-associated quantification problems in a concentration dependent manner, posing thus certain complications in cfNAs applications.…”

“…Despite several advantages, neither qPCR nor ddPCR is able to determine the size range of fragments, however, with certain modifications they may be used to estimate degradation status of the sample [ 285 , 286 ]. On the other hand, it is absolutely important to realize when designing and performing cfNAs analyzes that concentration estimates generally differ when using different methods, even for the same sample, mainly because of different principles and limitations of the methods, as well as because several factors may deteriorate results [ 268 ]. When comparing spectrophotometric, simple fluorometric (using PicoGreen) and qPCR-based (using SYBR Green I assay) quantification methods in the light of DNA fragmentation, although the overall sensitivity of the spectrophotometric measurement was found to be the lowest, this was the only method remaining unaffected by fragmentation of the measured fragments.…”

Extraction RNA Protocol from Blood and Testis Tissue of Local Roosters

“…The results of a comparative study with (Jeon et al, 2019), that the efficiency of methods for measuring the purity and concentration of nucleic acids in the (Quantus Fluorometry) method has more accurate quantitative estimates than the (TapeStation) method and that the measurement of the concentration of nucleic acids by (Quantus) depends on the intensity of the fluorescent dye flashes that are directly related to the nucleic acid. The device absorbs dissolved materials with wavelengths ranging from 160-900 nm.…”