ACTA Scientific Cancer Biology
Volume 2 Issue 1 January 2018
Quantity and quality of circulating cell-free DNA (cfDNA) from plasma is highly variable, with frequent contamination of larger, genomic DNA as a consequence of hemolysis during blood processing. Due to the inherent variability of cfDNA, it is imperative to implement a pre-analytical DNA quality check to reliably assess the amount of high-quality DNA in the sample, assuring the success of downstream high-cost analyses such as next-generation sequencing (NGS).
DNA was purified from blood, formalin-fixed paraffin-embedded (FFPE) tissue, buffy coat, and plasma samples using multiple methods. A multiplexed qPCR assay that included three different amplicon sizes (75-, 150-, and 300-bp) was employed for quality measurement of amplifiability, degradation, and genomic DNA (gDNA) contamination. The quantitative differences were de-termined and calculated as 75/300-bp ratios to assess DNA quality. Since gDNA is expected to be relatively larger in size, the ratio of 75-bp to 300-bp targets is indicative of the ratio of cfDNA to gDNA.
As expected, the 75/300-bp ratios of genomic DNA from various sources were around 1. Plasma cfDNA samples with low 75/300-bp ratios (< 10) are indicative of gDNA contamination, whereas samples with ratios of 10-100 are considered very clean, and ratios greater than 100 are indicative of degradation. The high-purity of cfDNA prepared using LIFE (Liquid Isolation-Free En-richment) technology was demonstrated by comparison of 75/300-bp ratios with cfDNA extracted by industry-leading Qiagen kit (ranges: 10.14 – 39.49 vs. 1.43 – 2.65). FFPE DNA qualities were highly variable with ratios ranging from 5.42 to 156.93, consistent with the notion that they are probably fragmented, damaged, and even degraded.
Data derived from the multi-size target qPCR assay on multiple sample types confirmed the outstanding performance of our LIFE technology over Qiagen extraction method in both cfDNA quality and quantity. Using our innovative cfDNA sample prepa-ration coupled with advanced technology for difficult samples like plasma can ensure getting the most clinically relevant information out of liquid biopsy, thus saving time, cost, and preventing loss of information from precious specimens.
Keywords: Cell-Free DNA; Isolation-Free; Enrichment; Liquid Biopsy