Plasma DNA Puzzle

whw · 06-27-2011, 11:57 AM

Hi All

I'm trying to isolate DNA from large volumes of plasma (anywhere from 10 to 50mL) in order to sequence it. I've been using Norgen's Circulating DNA Isolation kit but my yields have been very low so far, about 1.8ng of DNA per 5mL of plasma. Does anyone have any experience with either the Norgen kit or isolating nucleic acid from such large volumes?

Thanks!

genlyai · 07-13-2011, 08:11 AM

I second this question. Would love to hear of any experience on the board.

MattEwert3 · 07-28-2011, 10:42 PM

I am a scientist who has developed and clinically tested a large volume plasma sample prep system (5 - 18 ml) as part of a sepsis study. My method provides a higher yield and purity than all others we have tested.

The difference is obvious.

My email address is mewert@imigene.com

This is not a sales pitch. We have learned much about plasma prep as part of a US Army funded project and wish to share the discovery.

ECO · 07-28-2011, 10:46 PM

Quote:

Originally Posted by MattEwert3

This is not a sales pitch.

Then post the method.

MattEwert3 · 07-28-2011, 11:02 PM

Sorry to have breached protocol. While I am new to your forum I have been at this plasma project for some time, spent loads of cash but published none of it yet.

I just saw the request for improvements and know that we need more than the few hundred samples, which have already been tested, in order to be confident of the biochemistry we are proposing relative to nucleic acid extraction from plasma.

Since I have been so busy with other matters, your forum seemed an easy way to examine potential scientific collaboration. My company is for profit however and if my request for contact is frowned upon, please accept my appology.

ECO · 07-29-2011, 09:47 AM

I don't mind either way, just be up front about your intentions as "share" and "sell" are different things.

There is no shame in commercializing your technology, as I'm sure there are quite a few people interested in an improved method.

But you're correct, typically we keep overtly commercial posts in the Vendor Forum. If you want to start a thread there specifically soliciting collaborations for your plasma DNA extraction method I will be happy to give your account permissions to post there.

Thanks for understanding!

shawpa · 08-02-2011, 06:29 AM

I suggest you look for the following article on pubmed."Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus." I believe this method was pioneered by Lo YM and is used in most of his papers. This is the method that we use. I would post the method but I am not sure if you get in trouble by not citing this. I don't know what your source is but we get anywhere from 0.3-1.5ng(if we are lucky) of DNA per ml of plasma. Lo says you get much more but extensive testing on our side doesn't back this up. Most of the time we don't quantify it because it is not necessary for our use but to do so you need to either do high sensitivity bioanalyzer or use qPCR methods.

whw · 08-02-2011, 09:57 AM

Thanks for the tip shawpa, I've seen a lot of papers using the QIAamp kit but have been trying to steer clear of it because of the low input volume. The addition of a speedvac helps but I'm not sure I can use that method because I have infectious samples.

I'm interested in deep sequencing the circulating DNA (illumina platform) so would like a) avoid any PCR before my libraries are prepped and b) maximize the amount of DNA I have to start with. Have you made any libraries from your samples?

I've also read about differences in handling samples, some papers suggest EDTA and prompt DNA isolation to avoid sample degradation and contamination with genomic DNA as cells lyse. Some even treated with formaldehyde to stabilize DNA/prevent cell lysis. Do you have anything experience with these issues?

Thanks again,
WHW

shawpa · 08-02-2011, 10:11 AM

Making illumina libraries is what I end up doing with my samples. Not sure what you mean by "low input volume". Do you mean that you have too much plasma that you need to process. You can reload the columns to a point. I do PCR my samples eventually according to the protocol. As for sample prep. As soon as we get the blood, we separate it and aliquot it just like the protocol says. Then store in -80 till use.

whw · 08-03-2011, 11:41 AM

low input = how much plasma you can load onto the column, just like you said. I would like to load more than 200uL in order to get the most DNA possible. anyway thanks again for the paper, those fetal DNA guys seem to know what theyre doing.

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06-27-2011, 11:57 AM	#1
whw Member Location: Bethesda MD Join Date: Jun 2011 Posts: 19	Plasma DNA Puzzle Hi All I'm trying to isolate DNA from large volumes of plasma (anywhere from 10 to 50mL) in order to sequence it. I've been using Norgen's Circulating DNA Isolation kit but my yields have been very low so far, about 1.8ng of DNA per 5mL of plasma. Does anyone have any experience with either the Norgen kit or isolating nucleic acid from such large volumes? Thanks!

07-28-2011, 10:42 PM	#3
MattEwert3 Junior Member Location: USA Join Date: Jul 2011 Posts: 2	Large Volume Plasma I am a scientist who has developed and clinically tested a large volume plasma sample prep system (5 - 18 ml) as part of a sepsis study. My method provides a higher yield and purity than all others we have tested. The difference is obvious. My email address is mewert@imigene.com This is not a sales pitch. We have learned much about plasma prep as part of a US Army funded project and wish to share the discovery.

07-28-2011, 11:02 PM	#5
MattEwert3 Junior Member Location: USA Join Date: Jul 2011 Posts: 2	Plasma Prep Response Sorry to have breached protocol. While I am new to your forum I have been at this plasma project for some time, spent loads of cash but published none of it yet. I just saw the request for improvements and know that we need more than the few hundred samples, which have already been tested, in order to be confident of the biochemistry we are proposing relative to nucleic acid extraction from plasma. Since I have been so busy with other matters, your forum seemed an easy way to examine potential scientific collaboration. My company is for profit however and if my request for contact is frowned upon, please accept my appology.

07-13-2011, 08:11 AM	#2
genlyai Member Location: Boston, MA Join Date: Aug 2009 Posts: 39	I second this question. Would love to hear of any experience on the board.

07-29-2011, 09:47 AM	#6
ECO --Site Admin-- Location: SF Bay Area, CA, USA Join Date: Oct 2007 Posts: 1,358	I don't mind either way, just be up front about your intentions as "share" and "sell" are different things. There is no shame in commercializing your technology, as I'm sure there are quite a few people interested in an improved method. But you're correct, typically we keep overtly commercial posts in the Vendor Forum. If you want to start a thread there specifically soliciting collaborations for your plasma DNA extraction method I will be happy to give your account permissions to post there. Thanks for understanding!

08-02-2011, 06:29 AM	#7
shawpa Member Location: Pittsburgh Join Date: Aug 2011 Posts: 72	I suggest you look for the following article on pubmed."Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus." I believe this method was pioneered by Lo YM and is used in most of his papers. This is the method that we use. I would post the method but I am not sure if you get in trouble by not citing this. I don't know what your source is but we get anywhere from 0.3-1.5ng(if we are lucky) of DNA per ml of plasma. Lo says you get much more but extensive testing on our side doesn't back this up. Most of the time we don't quantify it because it is not necessary for our use but to do so you need to either do high sensitivity bioanalyzer or use qPCR methods.

08-02-2011, 09:57 AM	#8
whw Member Location: Bethesda MD Join Date: Jun 2011 Posts: 19	Thanks for the tip shawpa, I've seen a lot of papers using the QIAamp kit but have been trying to steer clear of it because of the low input volume. The addition of a speedvac helps but I'm not sure I can use that method because I have infectious samples. I'm interested in deep sequencing the circulating DNA (illumina platform) so would like a) avoid any PCR before my libraries are prepped and b) maximize the amount of DNA I have to start with. Have you made any libraries from your samples? I've also read about differences in handling samples, some papers suggest EDTA and prompt DNA isolation to avoid sample degradation and contamination with genomic DNA as cells lyse. Some even treated with formaldehyde to stabilize DNA/prevent cell lysis. Do you have anything experience with these issues? Thanks again, WHW

08-02-2011, 10:11 AM	#9
shawpa Member Location: Pittsburgh Join Date: Aug 2011 Posts: 72	Making illumina libraries is what I end up doing with my samples. Not sure what you mean by "low input volume". Do you mean that you have too much plasma that you need to process. You can reload the columns to a point. I do PCR my samples eventually according to the protocol. As for sample prep. As soon as we get the blood, we separate it and aliquot it just like the protocol says. Then store in -80 till use.

08-03-2011, 11:41 AM	#10
whw Member Location: Bethesda MD Join Date: Jun 2011 Posts: 19	low input = how much plasma you can load onto the column, just like you said. I would like to load more than 200uL in order to get the most DNA possible. anyway thanks again for the paper, those fetal DNA guys seem to know what theyre doing.