Optimizing the yield and utility of circulating cell-free DNA from plasma and serum
Introduction
Cell-free circulating DNA (CFDNA) has been studied in a wide range of physiological and pathological conditions, including pregnancy, trauma, inflammatory disorders and malignancy [1], [2]. It is present in normal healthy individuals at low concentrations (ng/ml) [3]. Raised levels of CFDNA in cancer patients have been reported in many tumour types [2], [3], [4], [5]. The finding that genetic and epigenetic changes typical of tumours can be detected in CFDNA from cancer patients, suggests that the excess CFDNA is of tumour origin. Although the precise mechanism of DNA release into the blood remains uncertain, it probably derives from a combination of apoptosis, necrosis and active release from tumour cells [6]. Such cell-free DNA has shown promise for improving early clinical diagnosis, prognostication and disease monitoring in inaccessible tumour types, such as lung cancer [7], [8], [9], [10], [11].
Although higher levels of CFDNA are consistently reported in cancer patients than healthy controls, there is considerable variation between studies. This may be attributable to differences in study design including selection of patient and control groups, and the methods used to extract and quantify CFDNA [10], [12], [13], [14]. No agreed standards exist, and many publications fail to specify how samples were obtained, processed and analyzed. The problems of prolonged sample storage have been highlighted by a recent report that DNA levels in plasma stored at − 80 °C declined by 30% per year [15]. Thus, this study was designed to evaluate the factors most likely to influence the yield of CFDNA from clinical samples.
Various methods have been used to purify CFDNA, including using modified salting-out [16], chromatography resins [16], [17], magnetic beads [18], or guanidium thiocyanate [19]. The most popular is the QIAamp blood kit, which binds DNA to a silica-gel membrane, providing a fast and easy way to purify total DNA for polymerase chain reaction (PCR) analysis. We found that the recovery of CFDNA was less than 20% using the QIAamp DNA Midi Kit. Some authors have recommended using a predigestive buffer in plasma/serum samples to improve the results [16], [18], [20]. Therefore, we have evaluated standard and modified QIAamp protocols and compared them with a simple Triton/Heat/Phenol (THP) protocol. Because DNA is present in plasma/serum at such low concentrations (ng/ml), it is crucial to optimize laboratory protocols for the processing and extraction of CFDNA.
Section snippets
Sample collection and preparation
To compare different CFDNA extraction protocols, commercial pooled human serum (SLI Ltd, UK) was spiked with reference genomic DNA (Sigma-Aldrich, UK) or a known amount of linearized bcl-2 plasmid reference DNA (final concentration in serum: 0, 50 and 100 ng/ml). For clinical samples, 20 ml peripheral blood was collected from healthy volunteers into EDTA tubes [21]. Plasma was separated by double centrifugation (800 g for 10 min, separation, and 1600 g for 10 min). Plasma aliquots were
Optimization of QIAamp protocol
Digesting or denaturing the plasma/serum proteins is one of the most important stages during the extraction of CFDNA. We compared the protease provided in the QIAamp Blood Kit with proteinase K for the purification of low quantities of CFDNA. Fig. 1 shows that pre-incubation of serum samples with proteinase K (400 μg/ml) at 37 °C for 1 h (Q37-1, P37-1) significantly increased the DNA recovery compared to no incubation (QN, PN) (QN vs Q37-1: p = 0.003; PN vs P37-1: p = 0.002) and there was
Discussion
Published studies on CFDNA in cancer patients give remarkably little detail about the conditions under which clinical samples were obtained, transported, stored or processed, making comparisons of the reported results difficult. Very few studies have addressed the efficiency or reproducibility of their chosen DNA extraction methods. We were initially surprised to obtain DNA yields of less than 20%, but a detailed literature review showed that this was not unusual [16], [17]. There is clearly a
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