decreasing 230/260 Ratio

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Questioner25.08
Questioner25.08's picture
decreasing 230/260 Ratio

I´ve isolated DNA from FFPE Tissue using the QIAamp DNA FFPE Tissue Kit, using water instead of ATE Buffer. In first measurement after DNA-isolation with ND1000 the results were good. I`ve stored the DNA at 4°C. In a remeasurement some days later the 230/260 ratio was not as good as in the first measurement and decreased further in the following weeks. Examples 230/260: 2,03 to 1,58; 2,17 to 1,63 in 20 days.
Anyone who has an idea? Somebody suggested the water may have evaporated leaving relatively more salt in the tubes, but samples isolated with same Kit, stored in same tubes did not show this problem.
Thanks

Ivan Delgado
Ivan Delgado's picture
To begin with DNA extracted

To begin with DNA extracted from FFPE tissue is by nature inherently not of the best quality (FFPE is known to degrade DNA), so even if you are able to extract DNA from FFPE tissue I would be very careful in the way I store this DNA.

By eluting your DNA in water, and then storing it at 4oC, you are essentially exposing your DNA to degradation. Water is by far not the best solution to resuspend DNA. While it is ok to elute with DNA for a number of reasons and applications, it is highly recommended to use it right away or store it frozen if you have to. Storing DNA at 4oC in water will result in DNA degradation almost all the time.

And DNA degradation would explain the lower 230/260 ratios.

My two cents.

Questioner25.08
Questioner25.08's picture
thanks, but my problem is

thanks, but my problem is that this method is commonly used in our instiute and i`ve tested samples of my collegues (isolatedt with same kit, eluated with water, stored in the same tubes) and did not find decreasing 230/260 ratios. And i found nobody who had heard of DECCREASING ratios. Bad, yes, but not decreasinng.
I thought the idea of evaporation combined with acid hydrolysis sounds convincing, but then the problem would have to occur more often?
Maybe something was wrong with the charge of tubes i used. Or the water?

Ivan Delgado
Ivan Delgado's picture
I would argue that just

I would argue that just because it is a method that is commonly used does not preclude the possibility that in this particular instance you got DNA degradation (or some other problem that lowers the ratio).

Since this is a protocol that works all the time as you pointed out, then it is also possible that the samples in question may not have been that good and as a result you got this ratio problem.

I don't think evaporation and acid hydrolysis are the culprits, but they may. Either way I would get new samples and try again.