Validating quantitative PCR assays for cfDNA detection without DNA extraction in exercising SLE patients

Abstract

Circulating cell-free DNA (cfDNA) has been investigated as a screening tool for many diseases. To avoid expensive and time-consuming DNA isolation, direct quantification PCR assays can be established. However, rigorous validation is required to provide reliable data in the clinical and non-clinical context. Considering the International Organization for Standardization, as well as bioanalytical method validation guidelines, we provide a comprehensive procedure to validate assays for cfDNA quantification from blood plasma without DNA isolation. A 90 and 222 bp assay was validated to study the kinetics of cfDNA after exercise in patients with systemic lupus erythematosus (SLE). The assays showed ultra-low limit of quantification (LOQ) with 0.47 and 0.69 ng/ml, repeatability ≤ 11.6% (95% CI 8.1–20.3), and intermediate precision ≤ 12.1% (95% CI 9.2–17.7). Incurred sample reanalysis confirmed the precision of the procedure. The additional consideration of pre-analytical factors shows that centrifugation speed and temperature do not change cfDNA concentrations. In SLE patients cfDNA increases ~ twofold after a walking exercise, normalizing after 60 min of rest. The established assays allow reliable and cost-efficient quantification of cfDNA in minute amounts of plasma in the clinical setting. Additionally, the assay can be used as a tool to determine the impact of pre-analytical factors and validate cfDNA quantity and quality of isolated samples.

Physical activity specifically evokes release of cell-free DNA from granulocytes thereby affecting liquid biopsy

“…1 A). Typically, healthy individuals show higher cfDNA elevations in response to exercise compared to patients, which is likely associated with exercise duration and intensity [ 9 , 17 ]. However, Tug et al studied cfDNA kinetics in hematopoietic stem cell transplantation patients showing that short-term treadmill exercise increased cfDNA levels up to sixfold, with a mean of ~ threefold [ 9 ].…”

“…The cfDNA concentration was measured applying a pre-validated qPCR assay described in Neuberger et al [ 17 ]. For pyrosequencing 4 µl of the bisulfite converted DNA were amplified with region-specific biotinylated/unmodified primer pairs (see Additional file 2 ) using the PyroMark PCR kit (Qiagen) according to the manufacturer’s instructions using the following protocol: Initial activation at 95 °C for 15 min, 45 cycles of 30 s at 94 °C, 30 s at 56 °C, and 30 s at 72 °C followed by a final extension at 72 °C for 10 min.…”

The Salzburg 10/7 HIIT shock cycle study: the effects of a 7-day high-intensity interval training shock microcycle with or without additional low-intensity training on endurance performance, well-being, stress and recovery in endurance trained athletes—study protocol of a randomized controlled trial

“…Both ferritin and transferrin concentrations will be determined using human enzyme-linked immunosorbent assay kits (Promocell and Invitrogen). Venous cfDNA will be quantified by analyzing unpurified plasma via quantitative real-time PCR (qPCR) as described elsewhere [ 45 ]. Circulating miRNAs will be isolated out of plasma samples (Macherey-Nagel and Qiagen) and certain miRNAs which have previously been shown to change in response to endurance exercise will be quantified via qPCR as previously described [ 46 ].…”

Feasibility of Cell-Free DNA Measurement from the Earlobe during Physiological Exercise Testing

“…Concentrations of cfDNA were quantified by analysis of unpurified plasma by quantitative real-time PCR (qPCR) as described elsewhere [ 22 ]. Briefly, plasma was diluted (1:10 in H 2 O) and was used as template for qPCR.…”

“…Ahead of the measurements, the linearity, limit of quantification, and limit of detection of the assay were established [ 22 ]. In addition, a set of two reference samples were pre-validated and were included in each run.…”