We have some RNA that was extracted using the Zymo directzol RNA extraction kit. We used trizol and then did a chloroform phase separation. We see really poor 260/280 and 260/230 ratios. It was suggested that I try re-running the RNA through the extraction to clean it up. Our cleanup kit did not really make a difference. Maybe it's possible that we have a lot of matrix or glycogens binding to the samples.
Our RNA volume is 10uL, so I was just trying to figure out the logistics of how to increase the volume to run it through the extraction again. Would I just need to increase the volume with water and add equal amounts of ethanol? Has anyone had success doing this?
Thank you,
Merissa
I usually do 2 chloroform phase extractions before adding to the directzol RNA columns. I know that the protocol says to start directly from trizol. I tried and I think that's BS.
You lose a lot of RNA if you RNA it through columns again and honestly in any "re-precipitation" protocols.
Are you doing from tissues or cells from cultures? If it's from tissues, I recommend, re-extracting using the double chloroform I mentioned. And DO NOT homogenize your cells with sonication. I found that damages RNA as well. I recommend mechanical homogenization using mortar and pestle or those spinning homogenizer.
I normally use Zymo research RNA clean and concentrator-5 kit. And it works for me fine. I would have a look at the homogenization of the samples. And while column separation I use an equal volume of ethanol for total RNA separation. Which means, 360ul of the aqueous phase and 360ul of ethanol.
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