cfDNA Recovery and EGFR Mutation Detection in Preserved Plasma
60% Higher cfDNA Recovery from Preserved Plasma with NEAT Technology - NEAT cfDNA Extraction Kit delivers higher cfDNA recovery, improved purity, and increased EGFR mutation copy recovery following extended blood sample storage.
Application: Liquid Biopsy | cfDNA Extraction | EGFR Mutation Detection
Technology: Nanotrap® Extraction Advanced Technology (NEAT)
Product: NEAT cfDNA Extraction Kit (SKU 77300)
Source: Ceres Nanosciences Application Note #108 — cfDNA Recovery and EGFR Mutation Detection in Preserved Plasma
Overview
Reliable cell-free DNA (cfDNA) recovery is critical for liquid biopsy applications, particularly when detecting low-frequency circulating tumor DNA (ctDNA) mutations. While preservative blood collection tubes extend the time available for sample processing, preservation chemistry and storage conditions can introduce challenges for downstream DNA extraction.
In this application note, researchers evaluated the performance of Ceres Nanosciences' NEAT cfDNA Extraction Kit with Norgen's cf-DNA/cf-RNA Preservative Tubes under challenging storage conditions.
The study compared NEAT with a commercially available column-based extraction kit, assessing cfDNA recovery, purity, and detection of two clinically relevant EGFR mutations.
NEAT recovered an average of 60% more cfDNA than the comparator kit from contrived plasma samples, maintained consistent cfDNA recovery after extended storage, and delivered higher recovered copy concentrations of EGFR L858R and T790M mutations.
Study Highlights
60% higher cfDNA recovery: NEAT recovered an average of 60% more cfDNA than the comparator extraction kit from plasma spiked with a ctDNA reference standard.
Improved cfDNA purity: NEAT produced eluates with a higher percentage of cfDNA relative to total extracted DNA, indicating reduced genomic DNA carryover.
Stable recovery after extended storage: cfDNA yields remained comparable to the Day 1 baseline after 30 days at 25°C and 8 days at 37°C.
Higher EGFR mutation copy recovery: Digital PCR analysis showed higher recovered copy concentrations of EGFR L858R and T790M mutations with NEAT than with the comparator kit.
Compatibility with preserved blood samples: The results support pairing NEAT with Norgen's cf-DNA/cf-RNA Preservative Tubes for workflows requiring extended sample storage before processing.
NEAT Technology in Action
Whole blood from two healthy donors was collected in Norgen cf-DNA/cf-RNA Preservative Tubes and evaluated under three conditions:
Day 1 following collection
Eight days at 37°C
Thirty days at 25°C
Following plasma separation, samples were extracted using either the NEAT cfDNA Extraction Kit with an automated KingFisher Apex workflow or a commercially available manual column-based extraction kit.
For mutation recovery analysis, plasma was spiked with Seraseq ctDNA Mutation Mix v2 at a 2% allele frequency. Extracted DNA was evaluated using the Agilent 4200 TapeStation system for cfDNA yield and purity, and the QIAGEN QIAcuity digital PCR system for EGFR L858R and T790M mutation detection.
The study assessed whether NEAT could maintain effective cfDNA extraction performance despite prolonged storage in chemically preserved blood samples.
Key Findings
Higher cfDNA Recovery and Purity
Across the contrived plasma samples, NEAT recovered an average of 60% more cfDNA than the comparator kit.
NEAT also produced consistently higher cfDNA purity, with cfDNA representing approximately 91–97% of total extracted DNA across the evaluated conditions, compared with approximately 82–91% for the comparator kit.
Higher recovery and purity can provide more usable cfDNA for downstream molecular analysis while reducing the contribution of unwanted genomic DNA.
Consistent Recovery After Extended Storage
NEAT maintained cfDNA recovery comparable to the Day 1 baseline after both extended storage conditions:
30 days at room temperature (25°C)
8 days at elevated temperature (37°C)
These findings support the compatibility of NEAT with Norgen's preservation chemistry, particularly for sample collection workflows where immediate processing may not be feasible.
Improved EGFR Mutation Recovery
Digital PCR confirmed detection of both EGFR L858R and T790M mutations in extracted plasma samples.
Across the tested conditions, NEAT eluates contained higher copy concentrations of both EGFR variants than the comparator kit, with statistically significant differences in several comparisons.
Greater recovery of mutation-containing DNA provides additional molecular input for downstream analysis, an important consideration when working with low-abundance ctDNA targets.
Significance for Liquid Biopsy Workflows
Liquid biopsy testing depends on recovering sufficient high-quality cfDNA from limited plasma samples. Loss of target DNA during extraction or contamination from genomic DNA can reduce the amount of useful material available for molecular testing.
These results demonstrate how NEAT technology can improve cfDNA recovery and purity while maintaining compatibility with preserved blood collection workflows.
The combination of higher cfDNA recovery, reduced genomic DNA carryover, and stable performance after extended storage is particularly relevant for:
Multisite clinical research studies
Centralized testing laboratories
Remote sample collection programs
Oncology biomarker research and assay development
The study used plasma from two healthy donors and characterized mutation standards rather than patient-derived tumor DNA. The findings establish analytical workflow compatibility and comparative extraction performance; additional clinical validation would be needed to establish performance in patient samples.
NEAT technology helps researchers recover more of the cfDNA that matters—supporting more effective downstream molecular analysis, even when immediate sample processing is not possible.
cfDNA Recovery and EGFR Mutation Detection in Preserved Plasma
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