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Old 06-14-2016, 03:49 AM   #1
pig_raffles
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Location: Hawaii, USA

Join Date: Feb 2012
Posts: 20
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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