Comparison of Methods of Isolation and Quantification of Cell Free DNA from Plasma of Patients with Breast Cancer.

Abstract

Objective
 Cell-free DNA (cfDNA) released in response to necrosis in cancer patients The objective of this study was to compare the efficiency of two commercial and two manual methods for cell-free DNA extraction. as well as to search for a method that is easy to extract the DNA from the plasma and cost-effective.
 Methods
 Plasma samples seven in number of patients of Breast Cancer was taken. We evaluated DNA quantity and its subsequent amplification obtained by four different cfDNA isolation methods; Modified Phenol Chloroform Isoamyl, Triton Heat Phenol, EpiQUik Circulating Cell-Free DNA isolation Easy kit” (EpiGentek) and “Nucleospin cfDNA kit”. Extracted DNA was quantified using Qubit and quantitative real-time PCR.
 Results
 Quantity of cf DNA varied between different extractions methods of a total of seven samples analyzed. The highest quantity was found from the samples extracted from the Nucleospin XS kit and the extraction efficiency was significantly higher in a pairwise comparison with the other three methods (p-value <0.0001). The concentration of the cfDNA obtained by all four methods was assessed on a Qubit fluorometer. The concentration was higher for the Nucleospin ?MPC?THP?Epiquik kit The qPCR values were consistently higher for Nucleospin XS as compared to all others. This indicates good amplifiability of Nucleospin XS
 Conclusion
 We tested four methods of cf DNA extraction. In our hands, Nucleospin XS gave the best yield and amplifiability. It is a quick and cost-effective method and sensitive for quantification of cfDNA on Real-time PCR. Therefore, it is highly recommended for clinical use of plasma as a liquid biopsy.