9 replies to this topic

[ramblingrat]

Hi everyone,

 

We have isolated DNA from buccal swabs for NGS. The yield on the nanodrop was ok, but up to 100fold lower upon picogreen measurement. 

I know that picogreen is more specific for DNA and we've had lower yields by about 50% routinely, but 100fold?! 

 

Can anyone advise why this could happen and how to isolate DNA from buccal swabs in a NGS-sufficient quality?

 

Thank you so much!

 

R.

 

[jerryshelly1]

What are your 260/280 values on the nanodrop? If it fluctuates a great deal from 1.8, it will be indicative of contamination (<1.8) or RNA (>1.8). Are you running your picogreen in doublets? It could be that your DNA is not resuspended 100%, causing you to have a lower than expected DNA concentration.

[ramblingrat]

Hi,

Many thanks for your reply, the 260/280 values of the DNA were fine, too. The picogreen was actually measured by our collaborator, who is quite experienced in picogreen measurement, and I'd trust they resuspend it.

The reason I am asking is that it's not a 2- or 3- fold but 100(!)fold differences.

I also once heard that certain DNA isolation reagents could interfere with picogreen, but don't know how I could find out about that.

Has anyone isolated DNA from buccal swabs for NGS?

 

Many thanks!

[jerryshelly1]

We have used the oragene method. Make sure you are both using the same units (silly I know).

[BMF]

Picogram should show less amount of DNA. Because it only shows dsDNA.

http://probes.invitr...pis/mp07581.pdf

[ramblingrat]

Hi,

 

Thanks again! Actually the units are the same and there is no chance to obtain another sample such as saliva . The only available are these buccal swabs unfortunately, and I would like to find out if there's any way to get sufficient DNA from such samples or if something is interfering with the measurement. Do you know anyone who did buccal swabs for NGS or a source where I could find out?

 

Many thanks!

 

R.

[jerryshelly1]

Physician researchers tend to use buccal swabs when the patient cannot come to clinic and the sample has to mailed from far away. Because it is a buccal swab, you are going to get a very small amount of DNA. Depending on what you want to do with the DNA, you may or may not have enough. If you are trying to validate a variant, you probably have enough. If you are trying to an NGS like exome sequencing, you will probably not have sufficient DNA. This is just speculation. I have no idea how much DNA your core requires.

 

I would triple check your protocol and make sure all your solutions are at the right pH and are not precipitating out. You mentioned you had good 260/280; what were the values? 

 

Maybe you can find a good source in here:

http://highwire.stan...1&x=-632&y=-389

[almost a doctor]

Can I ask what concentration range you are measuring?   The nanodrop limit of detection is 2ng/ul +/- 2, while if I remember correctly (haven't used in a while), picogreen can measure down to picogram level, hence the name (?). 

Sorry if this sounds obvious, but that'll explain a 100 fold difference, if you have small amounts I'll trust the picogreen better. 

 

just my 2 cents

[hobglobin]

I think the picogreen measurement's limits depend on the equipment you use finally, i.e. if it's a Qubit, Picoflour or any other fluorometer (this includes also the nanodrop if it's the 3330 model), or a qPCR since it is a dye. I'd try again using a new calibration curve. Also make sure that the DNA is as clean as possible, perhaps you can reduce then the difference.

[ramblingrat]

Hi guys,

 

Thanks for your efforts to help me, despite I can't find anything here I haven't tried or ruled out already. I'll ask a company who announces they can do it (DNA from buccal swabs for NGS), just thought maybe there is an idea how the company does it .

 

Best,

R.