Quantitative PCR-Based Optimization for Plasma DNA Quality Using Single- and Multi-Copy Reference Genes

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Improving circulating free DNA (cfDNA) purification is vital for accurate plasma DNA assessment.
• Leukocyte DNA contamination can skew results in cancer biomarker research.
• Quantitative analysis of housekeeping genes can serve as a quality indicator for cfDNA.

Purpose of the Study:

• To develop and assess quantitative PCR (qPCR) assays for evaluating plasma DNA quality.
• To use housekeeping genes (LINE-1 and TOP1) as indicators of DNA fragmentation and contamination.
• To optimize cfDNA purification methods for cancer biomarker research.

Main Methods:

• Selected LINE-1 (L1) and TOP1 genes for analysis.
• Designed primer pairs to amplify short and long DNA fragments.
• Utilized real-time quantitative PCR (qPCR) to determine amplicon copy numbers and calculate the small-to-large amplicon ratio (S/L).
• Evaluated the impact of storage time and temperature on cfDNA extraction from K2EDTA tubes.

Main Results:

• The S/L ratio correlated with DNA fragmentation levels, with TOP1 showing higher sensitivity.
• Plasma DNA extraction kit performance varied, with Qiagen yielding the highest S/L ratio.
• Storage conditions significantly affected cfDNA quality, with S/L ratios decreasing after 3 days, especially at room temperature.

Conclusions:

• Developed qPCR assays to quantify plasma DNA fragmentation using single- and multi-copy reference genes.
• The S/L amplicon copy number ratio is an effective measure of DNA fragmentation status.
• This assay serves as a valuable tool for assessing plasma DNA quality in research settings.