High Interspecimen Variability in Nucleic Acid Extraction Efficiency Necessitates the Use of Spike-In Control for Accurate qPCR-based Measurement of Plasma Cell-Free DNA Levels.

Abstract

To assess the interspecimen variability associated with plasma DNA extraction in order to provide insight regarding the necessity to use an exogenous spike-in control when measuring cell-free DNA (cfDNA) levels using quantitative polymerase chain reaction (qPCR). Plasma specimens were obtained from 8 healthy individuals, 20 patients with cardiovascular disease risk factors, and 54 patients diagnosed with acute stroke. Specimens were spiked with an exogenous oligonucleotide fragment, and total DNA was extracted via automated solid phase anion exchange. We determined recovery of the exogenous fragment via qPCR and used this information to calculate DNA extraction efficiency. Plasma DNA extraction efficiencies varied dramatically between specimens, ranging from 22.9% to 88.1%, with a coefficient of variance of 28.9%. No significant differences in DNA extraction efficiencies were observed between patient populations. We strongly recommend the use of an exogenous spike-in control to account for variance in plasma DNA extraction efficiency when assessing cell free DNA (cfDNA) levels by qPCR in future biomarker investigations.