The researchers in this paper extracted DNA from plasma using Quiagens QIAamp MinElute Virus Spin Kit. Is it because they expected the cfDNA to be fragmented and small? Cost? The virus extraction kit is $300, the cfDNA extraction kit is ~1,000.
paper: Detection of EGFR mutations in plasma DNA from lung cancer patients by mass spectrometry genotyping is predictive of tumor EGFR status and response to EGFR inhibitors
Hi,
I think, as already mentioned, the virus kit is useful for cfDNA isolation because of the highly fragmented cfDNA. The difference to the cfDNA kit in addition to the price is the usable serum volume. I think you can use up to 5ml for the cfDNA kit.
One remark to the statement: "most of the excess DNA is from tumor cells". I am not working with lung cancer, but I do not think that this is the case. At least in ovarian cancer (our experience) and CRC (Diehl publications) the tumor DNA is most often only a small part of the total cfDNA.
Hi Vera,
I would ask the corresponding author of this paper (Marc Ladanyi, MD, [email protected]), because they will have the answer you are looking for. I took a look to the paper and although I am not an expert in cancer research I will try to give you some clues in this issue. In the paper the authors stated "Plasma samples from patients with lung cancer contain much higher levels of DNA than plasma from cancer-free patients. Most of this excess circulating DNA is believed to be released from the dying lung cancer cells at primary or metastatic sites". Therefore this DNA is in degraded form (small fragments), and a kit like the one used in the paper is suitable for cell free DNA isolation. cfDNA kits are used for cell-free fetal DNA, which are a lot more expensive, and probably also suitable for this type of analysis.
Hi,
I think, as already mentioned, the virus kit is useful for cfDNA isolation because of the highly fragmented cfDNA. The difference to the cfDNA kit in addition to the price is the usable serum volume. I think you can use up to 5ml for the cfDNA kit.
One remark to the statement: "most of the excess DNA is from tumor cells". I am not working with lung cancer, but I do not think that this is the case. At least in ovarian cancer (our experience) and CRC (Diehl publications) the tumor DNA is most often only a small part of the total cfDNA.
Dear Vera
The cfDNA is an excellent source for prenatal diagnoses and cancer diagnoses. Yes, you right, the cfDNA is fragmented and it is including fragments range from~ 40bp to 1500bp. The cfDNA kit is designed for purifying cell free fetal DNA (cffDNA) from plasma and also from amniotic fluid because it is able to purify the cffDNA from maternal cfDNA based on difference in their base pair size for further information you ought to read patent papers ( US 2011/0130558 A1 & US 2012/0171675 A1). Anyway, I should tell you the key component for purifying fragmented DNA is adding carrier DNA/RNA during ethanol or isopropanol precipitation. In fact, recently, I designed a cffDNA purification kit from amniotic fluid based on salting out method without applying any commercial kits like the cfDNA kit or the virus extraction kit. In my opinion,if you would be able to apply this protocol it doesn't cost you an arm and a leg! I should mention again that the key component is DNA/RNA carrier and incubating ethanol precipitation step mix at -20 c for at least 18-24h. Ultimately you would gather sufficient amount cfDNA for your downstream analyses.
Best,
Hadi
Hello Vera,
as our company manufactures a wide range of different extraction kits, I might also shed some light onto this. Norman is correct - with a virus isolation kit smaller fragments can be isolated (which cfDNA is) compared to blood DNA exctraction kits which are also mentioned in the literature for cfDNA extraction.
As cfDNA appears in plasma at low concentration, maximum sample input as denoted in the handbook of the kit manufacturer should be used. Given the target sequence is just a small fraction within the cfDNA - like in tumor analysis, specialized kits are beneficial in order to further increase the detection sensitivity as these kits (often) can handle large sample volumes.
At this stage I do not want to go into further details as I'm a 'commercial guy' (but still with a scientific heart). However, in case you would like to get further information on solutions we provide, please feel free to get in touch with me.
Good luck for your work !
Ingo
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