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Old 07-17-2015, 01:36 PM   #1
Angelmass
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Default ctDNA isolation from plasma

Hey guys,

We're currently looking for alternative ctDNA extraction kits. We currently use QIAGENs QIAamp circulating nucleic acid kit for our plasma extractions (10ml plasma). It does currently seem like the more plasma we use as a source, the less ng ctDNA/ml plasma we're getting, and we're concerned with the filters getting clogged. Right now we're around 60ng / ml plasma when plasma volume <= 5ml, and about 10ng / ml plasma when more than 5ml plasma was used.

Anyway I was wondering what kind of alternatives are out there that are viable methods, and what kind of yields you are seeing in the lab.

DNA size is about 100-300bp via Tapestation. Yields analyzed with Qubit.
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Old 07-20-2015, 12:40 PM   #2
Bioo Scientific
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Bioo Scientific's NextPrep-Mag™ cfDNA Isolation Kit should be able to help you. It is designed for extracting cfDNA from human blood plasma for NGS library prep using a scalable magnetic bead-based format. Typical input plasma volumes range from 0.6 mL – 4 mL, although amounts of plasma outside this range are also compatible with the kit.
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Old 07-20-2015, 02:19 PM   #3
Angelmass
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How much outside this range? We are trying to isolate 10ml plasma at a time.
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Old 07-21-2015, 08:56 AM   #4
Bioo Scientific
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Isolating 5 mL at a time is achievable. For 10 mL you'll need to perform two cfDNA isolations from plasma.
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Old 10-10-2015, 07:18 AM   #5
han526
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Default plasma ctDNA

Have you found any suitable kit for your application? Aline Biosciences now has a magnetic beads based kit for plasma cell free DNA extraction. it can deal with large volume of plasma. 10 mL is not a problem.

Also, how accurate is the Qubit quantification of ctDNA based on your experience?


Quote:
Originally Posted by Angelmass View Post
Hey guys,

We're currently looking for alternative ctDNA extraction kits. We currently use QIAGENs QIAamp circulating nucleic acid kit for our plasma extractions (10ml plasma). It does currently seem like the more plasma we use as a source, the less ng ctDNA/ml plasma we're getting, and we're concerned with the filters getting clogged. Right now we're around 60ng / ml plasma when plasma volume <= 5ml, and about 10ng / ml plasma when more than 5ml plasma was used.

Anyway I was wondering what kind of alternatives are out there that are viable methods, and what kind of yields you are seeing in the lab.

DNA size is about 100-300bp via Tapestation. Yields analyzed with Qubit.
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Old 10-13-2015, 12:12 PM   #6
Angelmass
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Quote:
Originally Posted by han526 View Post
Have you found any suitable kit for your application? Aline Biosciences now has a magnetic beads based kit for plasma cell free DNA extraction. it can deal with large volume of plasma. 10 mL is not a problem.

Also, how accurate is the Qubit quantification of ctDNA based on your experience?
At the moment, we're sticking with QIAGEN kits simply because the contract that we have stipulates that we use the contractor's protocol for guaranteed results during clinical workflow.

As for the accuracy, that's difficult to say, as our QC cutoff is pretty low (12ng; 0.20ng/ul), and anything above that is concentration independent. So we use it more for a qualitative check rather than quantitative. As far as we can tell though we haven't had any issues due to concentration errors.
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