We recommend a newer, more robust version of this kit: EpiQuik Circulating Cell-Free DNA Isolation Easy Kit
The EpiQuik™ Circulating Cell-Free DNA Isolation Kit utilizes magnetic beads based size-fractionation technology to isolate circulating cell-free DNA (ccfDNA) from mono-nucleosomal and di-nucleosomal complexes in plasma/serum samples. The isolated ccfDNA can be directly used for real time-PCR and DNA library preparation suitable for next generation sequencing. The kit has the following advantages:
- Uses innovative magnetic bead based size-fractionation technology for selective isolation of circulating cell-free DNA from plasma/serum that is mainly 170 bps in size. The isolated DNA can be directly used for both qPCR and NGS DNA library preparation.
- Fast and straightforward procedure can be finished within 2 hours. No gels, columns or centrifugation is needed.
- Efficient removal of proteins, salts, nucleases, PCR inhibiting substances, and other impurities such as polysaccharides, polyphenols and lipids.
- Sensitive and efficient DNA capture enables successful isolation with high recovery (>80% of input monoucleosomal DNA), even when the quantities of starting material are limited (as low as 0.1 ml).
- Manual and automation friendly – Scalable for single tube or 96-well plate formats.
Genetic and epigenetic analysis of circulating cell-free DNA (ccfDNA) in plasma/serum or other body fluids provides unique opportunities for early detection of a wide range of clinical disorders such as cancer, autoimmune disease, infection and fetal disorders. It was demonstrated that ccfDNA of clinical importance occurs predominantly as fragments of approximately 170 bases from mononucleosomes with a smaller proportion as fragments of 360 bases from di-nucleosomes [1,2 ]. Such nucleosomal complexes are released into blood circulation during apoptotic cell death and will be increased under various pathological circumstances such as inflammation, pulmonary embolism, autoimmune disease, and cancer [3,4]. It is also shown that using ccfDNA from such nucleosomal complexes for genetic or epigenetic analysis provides better and more accurate identification of physiological and pathological status . There are several methods currently being used for ccfDNA isolation from plasma and serum. All of these methods are based on capture of DNA by silicone column binding or phenol-chloroform separation. The DNA isolated by these methods contains both ccfDNA and non-ccfDNA, which may affect the accuracy of downstream analysis. To address these problems, Epigentek offers the EpiQuik™ Circulating Cell-Free DNA Isolation Kit for ccfDNA isolation.
Principle & Procedure
The EpiQuik™ Circulating Cell-Free DNA Isolation Kit contains all components which have been optimized for the simple and rapid isolation of small size nucleosomal DNA from plasma/serum. The mono- and di-nucleosomal complexes are efficiently captured via size-fractionation magnetic beads (ccfDNA Capture Beads) by applying the beads to a magnetic field (EpiMag™ HT (96-Well) Magnetic Separator or similar). The captured nucleosomal DNA is then enzymatically released, and purified using MQ Binding Beads by simply washing the beads. The purified ccfDNA is then eluted from the beads for immediate use or storage.
Both fresh and frozen plasma/serum from various sources can be used. However, fresh plasma/serum will generally give higher DNA yields than frozen. Furthermore, frozen plasma/serum will lead to DNA loss of about 10% per year. The input volume of plasma/serum can be from 0.1-1 ml with the standard volume of 0.5 ml per sample. If serum sample is used, the serum should be prepared within 6 hours after blood draw, since lysis of peripheral blood lymphocytes may cause an artificial increase in the amount of DNA during serum separation.
Fig 4. High selectivity of specifically isolating small size ccfDNA (mononucleosomal DNA): Panel A: 500 ng of unspiked control polynucleosome (up to 2000 bps); Panel B: The 500 ng of the same polynucleosome spiked in 0.5 ml plasma; Panel C: 500 ng of mononucleosome spiked in 0.5 ml plasma; Panel D: 500 ng of polynucleosome and 500 ng of mononucleosome, which were simultaneously spiked into 0.5 ml plasma.
1. Jahr S et al: Cancer Res. 2001, 61: 1659-1665
2. Suzuki N et al: Clin Chim Acta. 2008, 387: 55-58
3. Holdenrieder S et al: Crit Rev Clin Lab Sci. 2009; 46: 1-24
4. Schwarzenbach H et al: Nat Rev Cancer. 2011; 11: 426–437
5. Chan KCA et al: Clinical Chem. 2004, 50: 88-92