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Old 06-14-2016, 02:49 AM   #1
pig_raffles
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Default Advice on DNA Extraction from RNAlater stored tissue

Hi,

I am attempting to extract DNA from RNAlater stored tissue samples for
downstream DNA methylation sequencing (RRBS).

I have tried several different extraction protocols: Qiagen DNeasy
blood & tissue, Promega Wizard, Phenol/Chloroform and a spooling
protocol from Thermo-Fisher specifically intended for this type of DNA
extraction (could not see any DNA spooling). I have also tried soaking the tissue in both 1xTE buffer and water prior to tissue lysis, all with little effect. All of these extraction protocols I followed-up with ethanol precipitation to clean the extracts further. I have even now tried Machery-Nagel nucleospin gDNA clean-up columns with not much improvement

Whilst I successfully extracted some relatively high quality DNA (when
visualized on a gel), yields were lower than expected. More worryingly
the 260/230 ratios were low (<1.2) suggesting that a lot of salts were
being co-extracted alongside DNA. My main concern here is that these
salts could impede enzyme action during restriction-ligation or PCR
leading to problems with reproducibility. Oddly occasional samples come out looking perfect when visualized on a nanodrop

Has anyone had some success extracting DNA from RNAlater stored tissue
and would be willing to share some tips/advice on how to carry these
extractions out successfully and cleanly?

Many thanks in advance!
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Old 06-14-2016, 05:03 AM   #2
ATϟGC
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Howdy,

Low A260/230 can be caused by many contaminants including guanidine salts, phenolics, glycogen or other carbohydrates. In my experience if it is pervasive among different isolation procedures it is likely a co-precipitant originating from the sample tissue itself. Polysaccharides are a common culprit in plant and mollusk tissues and are co-precipitated by the alcohols that are in nearly all commercial kit binding buffers. I have seen commercial column-based kits shear high-molecular weight DNA due to the contaminants.

It would be very helpful if you told us what species, or at least the general group of organisms your are working with. There are a few good home-brew options that I have found helpful for improving A260/230 but I would want to know the taxon before making a recommendation. If you think the RNAlater is the culprit you could soak the sample in PBS or another inert buffer an hour or so before extraction but I would suspect the problem is co-precipitation of something like carbohydrates, which will not be helped by soaking the tissue in such a buffer.

Here is thread you may have already consulted from pmiguel that I have found helpful for diagnosis:
http://seqanswers.com/forums/showthread.php?t=21280
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Old 06-14-2016, 05:27 AM   #3
pig_raffles
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Apologies for the lack of detail.

I am using fish liver tissue.

It is a tissue that I have never had too much problem carrying out extractions with before (for ethanol stored tissue). In fact the issue previously has always been too much DNA!

I have soaked the tissue prior to lysis in both water and 1 x TE, it did not seem to noticeably improve the results. Would you expect a better removal of guanidine salts with PBS?

I am pretty certain that it is co-precipitaiton of guanidine salts from the RNAlater that is the main cause of the low 260/230 ratio and also is keeping DNA bound and unaccessible - hence the low yields. Once when trying a spooling protocol (the ThermoFisher protocol to be exact), I got massive amounts of salt precipitating out. However I would be happy to be proved wrong!
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Old 06-15-2016, 08:01 AM   #4
ATϟGC
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Hi again,

If you have already tried soaking the samples then I doubt that PBS would help you out.

If the problem is indeed guanidine-HCl or other chaotropic salts, I expect that those would be originating from the binding and perhaps the first wash buffers of the kits. I say this because RNAlater is essentially a saturated solution of Ammonium Sulfate along with 25 mM Sodium Citrate and 10 mM EDTA with a pH adjusted to ~5.2 [http://www.google.com/patents/US6204375]. I do not know why this would be that much different from ETOH but perhaps someone else might know.

I've mostly extracted RNAlater preserved leaf extracts but I have a colleague extracting from RNAlater preserved fish gill tissues so I'll post an update when they get some NanoDrop results.

All that said, you could try additional washes with the final buffer and an extra spin to dry out your spin columns to get rid of any salts. You could also try to clean up your spin column-eluted DNA with some ampure or similar beads but I have had little to no luck getting better nanodrop ratios with a secondary clean-up.

If you want to try another extraction method with a different mechanism of DNA precipitation, I have had good results with glycogen and polysaccharide rich plant and shellfish tissues using the Xin and Chen method:

https://plantmethods.biomedcentral.c...1746-4811-8-26
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Old 06-20-2016, 12:43 AM   #5
pig_raffles
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Thanks for the reply,

It seems like you have a lot of experience carrying out DNA extractions from RNAlater, have you had a chance to compare the extractions for yield/quality to those of the same tissue, but say preserved in ethanol?

I am slightly worried by the possibilty that it is not the storage medium but the actual tissue itself that is the issue. However I think I would still get lots of DNA, but it wold be highly fragmented and of low quality if the tissue was the main issue

Your colleagues views would be greataly appreciated
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Old 06-27-2016, 10:08 AM   #6
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Hello again,

My experience with DNA extraction from RNAlater preserved tissues was mostly plant-related but I am about to do some extractions from RNAlater preserved fish gills so I have been worried about this myself.

A friend was doing some necropsies on 4 Salmo salar the other day and they kindly provided us with some gill tissues so we preserved them in either RNAlater or 100 ETOH for about 24 hours before starting tissue lysis.

I extracted gill tissue from each of the four fish that had been put into the ethanol, RNAlater or RNAlater followed by a one hour PBS bath. This gave a total of 12 gill tissue samples I modified the Xin and Chen method slightly by including proteinase K in the CTAB lysis buffer and made a few other minor modifications.

The tissues immersed in PBS turned into a slimy snot and they were hard to load into the lysis buffer. All the other treatments resulted in a snotty mess at some point of the extraction process (mostly after the chloroform wash but some final eluates had a small snot cloud in the elution buffer).

I ran all the 12 extracts on the nanodrop and the curves were surprisingly nice after seeing so much snot during the extraction. The ETOH samples gave the highest yields and best A260/230 (330 ng/ul, A260/230=2.27) compared to the RNAlater (168 ng/ul, A260/230=1.97) and RNAlater-PBS bathed samples (162 ng/ul, A260/230=1.99). My colleague extracted the same samples with the MN animal tissue kit and got the same general trend.

That is all I intend on doing in the near future with these samples but I'll let you know if my colleague does any PCR or agarose gel with these extracts.

goodluck
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Old 07-08-2016, 02:27 AM   #7
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Thanks again for the advice. This will be really useful for me and, I am sure, other people
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dna extraction, methylation, rnalater, rrbs

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