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Has anybody had any success in extracting cell free DNA from plasma without using expensive kits? I've already spent a lot of my own blood trying to use standard phenol/chloroform methods with very minimal yield. I know it's possible that since I am not pregnant and don't have cancer (I hope) my blood may not have a large quantity of cell free DNA to begin with but I'm afraid to start using precious experimental samples until I'm confident I have a technique that will work.

For some background, cell free DNA is DNA that is circulating in the plasma (as opposed to the majority of DNA which is cellular). This has become an exciting new way to analyze fetal DNA from a mother's blood without doing a dangerous amniocentesis. There's also strong evidence that tumour DNA can also be found in some abundance this way (most studies seem to be with prostate cancer).

Qiagen has a kit for sale to extract this DNA but it is pretty expensive and I would still think that phenol/chloroform would give me a high (if less pure) yield. There is a paper in Cancer Biomarkers 2013 by Mazurek et al that claims that compares methods and shows a reasonable yield with this method but I haven't reproduced it. Has anybody here had success and if so what modifications did you use? Is it just time to move on to using samples that are more likely to have larger amounts of cell free DNA?

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  • $\begingroup$ I know that if I inject plasmid DNA into a mouse tail vein with nothing to protect it, it's chopped up within 5 minutes. You sure the DNA is really naked DNA floating freely in the blood? There an awful lot of nucleases in there. $\endgroup$ – user137 May 1 '15 at 21:56
  • $\begingroup$ No, it is contained within microparticles or enveloped within a membrane as far as I understand but it is not cellular and can be found in plasma. $\endgroup$ – Jack May 1 '15 at 22:00
  • $\begingroup$ How about using a density gradient seperation to enrich your atarting material? Also, instead of precipitation, using silica microbeads to pull the dna after getting rid of the lipid membranes (in presence of appropriate nuclease inhibitor cocktail, of course!) might possibly increase your yield. $\endgroup$ – WesternBlöd May 1 '15 at 22:10
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    $\begingroup$ purpose of phenol/chlorophorm extraction, as I remember, is to precipitate/separate proteins, lipids and DNA. So if your dna is bound to proteins, you won't clear it $\endgroup$ – aaaaaa May 1 '15 at 22:11
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    $\begingroup$ Maybe do a proteinase K digestion before your phenol chloroform extraction. That would destroy the nucleases. If you extract first, you'd probably have some nuclease carry over and damage your freshly unprotected DNA. $\endgroup$ – user137 May 1 '15 at 23:28
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To note: I have no personal experience with this protocol. I am forwarding this paper, passed on to me by a colleague

There are many kits and methods available that don't rely on Qiagen. Or alternatively, if you ask them nicely, they do heavily discount their first kit you buy off them as a sort of trial (as they dont do samples).

However, to answer your primary question, are you adding a detergent and heat denaturing your samples first before phenol-chloroform (Triton/Heat/ Phenol)?

Blood samples for CFDNA analysis are collected into EDTA tubes and held at/or below RT (18 °C–22 °C) for no more than 2 h before separation of plasma. Plasma should be separated from whole blood samples by double centrifugation (800 g and 1600 g for 10 min, separately), avoiding leukocyte lysis. We prefer to isolate CFDNA immediately after plasma separation to minimize the effect of prolonged storage. Otherwise, samples should be aliquoted into small portions and stored at −70 °C before extraction because fragmentation of CFDNA might be caused by repeated freeze–thaw cycles

For the THP method, 500 μl of plasma/serum was mixed with 5 μl Triton X-100 (Sigma-Aldrich, UK) and heat denatured at 98 °C for 5 min. Samples were placed on ice for 5 min, then extracted with an equal volume of phenol–chloroform–isoamyl alcohol (25:24:1, v:v:v) (Sigma-Aldrich, UK) and centrifuged for 10 min at 14,000 g. The aqueous phase was precipitated overnight with 1/10 volume of 3 M NaOAc and 2.5 volume of 100% ethanol at −20 °C. The DNA pellet was washed with ethanol, air-dried and resuspended in 50 μl of ddH2O.

In the THP protocol, 98 °C incubation was used to inactivate PCR inhibitors and denature proteins in plasma/serum samples. Different times and temperatures were tested before choosing this. Since we had noticed substantial DNA contamination in SDS, Triton X-100 was used in the protein solubilization step. Direct conventional PCR was performed successfully on heat-denatured samples and able to detect 25 ng/ml DNA, indicating that this simple procedure offered sufficient protein digestion and inhibitor elimination for PCR analysis. The THP protocol gave a significantly higher yield of pure DNA solution for qPCR analysis than the QIAamp kit. The THP protocol showed high efficiency even with small DNA fragments as low as 100 bp.

You might want to read this: Xue, Xiaoyan, et al. "Optimizing the yield and utility of circulating cell-free DNA from plasma and serum." Clinica Chimica Acta 404.2 (2009): 100-104.

A quick search of google scholar also revealed a few other papers you might be interested in:

Fleischhacker, Michael, et al. "Methods for isolation of cell-free plasma DNA strongly affect DNA yield." Clinica Chimica Acta 412.23 (2011): 2085-2088.

UPDATE:

After reading this (Fong, Siew Lee, et al. "Comparison of 7 methods for extracting cell-free DNA from serum samples of colorectal cancer patients." Clinical chemistry 55.3 (2009): 587-589.) which gives about 7 protocols (sadly without any references for some reason) I went hunting for came up with this:

DNA was extracted from 1.0 ml plasma by phenol-chloroform method with slight modifications. One ml of plasma was treated with 1x SDS/Proteinase K solution (0.5 mg/ml) (1:1), incubated overnight at 56 °C followed by phenol-chloroform (4:1) treatment and centrifuged at 7 000 rpm. The upper layer was transferred into fresh 15 ml centrifuge tubes and the same step was repeated again; DNA was precipitated by adding glycogen (0.1 µg/µl), ammonium acetate (7.5 M) and absolute alcohol. DNA pellet was obtained by centrifuging at 7 000 rpm; was washed with 70% alcohol at 10 000 rpm, dried and finally dissolved in TE buffer at 56 °C and stored at -20 °C until further use.

You can read about the above method here: Singh, Deepika Misra1 Renu, and Monisha Banerjee. "A simple and reliable method of obtaining fetal DNA from maternal circulation; its accuracy and sensitivity."

Also, if its available to you, it might be worth it:

Gahan, Peter B., ed. Circulating Nucleic Acids in Early Diagnosis, Prognosis and Treatment Monitoring: An Introduction. Vol. 5. Springer, 2014.

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  • $\begingroup$ Thanks for this! I have used essentially that second method except precipitated DNA with KCl instead of ammonium acetate... can't imagine that makes much of a difference. I also used SDS/Proteinase K but at a lower temperature. Because of my workflow I am unable to extract the DNA right away so I store the plasma at -80. I think the big thing though is that all of these papers use plasma that they expect will have cfDNA in it whereas I'm using control plasma so that may have the biggest impact on my yield I'm starting to suspect. $\endgroup$ – Jack May 2 '15 at 1:18
  • $\begingroup$ What temp is your proteinase K reaction? I've only ever seen it at 50 - 60 C. $\endgroup$ – user137 May 2 '15 at 5:25

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