Chimeric External Control to Quantify Cell Free DNA in Plasma Samples by Real Time PCR

Area of Science:

• Molecular Biology
• Biochemistry
• Genomics

Background:

• DNA isolation methods significantly impact real time PCR quantification, especially for cell-free DNA (cfDNA).
• Standardizing cfDNA extraction efficiency, yield linearity, inhibitor presence, and fragment size is challenging.
• An External DNA Control (EDC) using chimeric sequences was developed for qPCR standardization.

Purpose of the Study:

• To develop and validate an External DNA Control (EDC) for standardizing quantitative PCR (qPCR) of cfDNA.
• To assess the impact of DNA extraction procedures on cfDNA quantification.
• To monitor the stability of cfDNA concentration in blood samples over time.

Main Methods:

• A 167 bp chimeric DNA fragment (EDC) from parvovirus B19 and pBHA was designed.
• Plasma samples from normal subjects were treated and processed for DNA extraction.
• The EDC fragment was spiked into plasma samples, followed by DNA extraction and qPCR analysis.

Main Results:

• Real time PCR revealed decreased cfDNA quantification in untreated samples compared to treated ones.
• The EDC fragment's threshold cycle (Ct) remained constant across treated and untreated samples.
• This indicates that observed cfDNA Ct value differences were due to sample treatments, not extraction variability.

Conclusions:

• Spiking cfDNA-sized fragments into plasma minimizes bias from sample preparation and extraction.
• Using a standard External DNA Control is highly recommended for accurate cfDNA extraction and quantification.
• This approach enhances the reliability of cfDNA data analysis.