I'm looking for circulating cell-free DNA isolation from either serum or plasma. After isolation I will perform Bisulfite conversion to these DNAs, so I need maximum amount. However there are lots of different kits and protocols online. Which kit/protocol has the maximum yield?
We've been using Qiagens QiaAmp circulating nucleic acid kit and NEB Maxwell circulating DNA kit quite extensively. In the end we stuck with the maxwell, not only for convenience but in our hands the quality seemed much better (measured with tape station). But I think Qiagen also launched a new kit we haven't tried yet.
In general try to use as much blood/plasma as possible, to maximize yield. There is huge patient to patient variance in the yield. Sample handling is also and issue - you don't want the dead or dying buffy coat cells to contribute/dilute your cfDNA. For transport or otherwise delayed plasma separation Streck tubes may be an option.
Hope that helps.
Thank you for your answer. I am currently trying QIAamp DNA Blood Mini Kit to isolate DNA from 200ul human serum. I have read that we can increase DNA yield by adding carrier RNA/DNA to the sample. Have you ever perform such a step/what is your opinion?
Wow, 200µl seems very little. We are currently using 5ml of plasma and our yield is btw. 60pg and 30ng in total (100-400bp fraction). Yes, huge variation, and sadly enough the 30ng is more of an outlier than the 60pg. We are using this for NGS, so you might make do with less than we need. We've never added carrier NAs, it sounds interesting, though. However, if you have the chance try to increase your sample volume, that's your best chance. For establishing this method you can use yours or your colleagues as healthy donors, and see where you end up yield-wise with increasing volumes of blood/serum.
Good luck!
One last question: I measure my DNAs with NanoDrop, in dsDNA setup. However I have read that we can also use Qubit. How do you measure your isolated DNAs?
Hi Sedef,
In my hands, the QIAMP midi kit from QIAGEN works perfectly, with relative high amounts of cfDNA in plasma. I always quantify this with the QUBIT HS kit because it is the one with the highest sensitivity.
Thanks and my best regards,
Enrique
Hello Mr. Enrique,
Can the QIAMP Midi kit be used to isolate cell free DNA from frozen plasma samples right away? Or, does this kit require processing of blood first? The protocol states DNA isolation from whole blood. I am a little confused about this.
There are some suggestions that you can use plasma directly in the PCR. This paper provide some info about it. I think worth trying this protocol once. http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0087838#pone-0087838-t001
Dear Sedef,
did you manage to do some methylation analysis? Which kit did you use for bisulphite conversion, adn how long were the primers you used? We are planning such experiment as well, and would be happy to hear from you, how did you do it.
Best,
Alja
Dear Alja,
I used Zymo Research's EZ DNA Methylation-Lightning Kit for bisulfite conversion. Our forward primer was 28 and reverse primer was 25 bp long. However due to integrity issues, I performed nested-pcr.
Best
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