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RNA Freeze/Thaw and NanoDrop Concentration


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#1 Relaxosome

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Posted 13 February 2014 - 09:14 PM

Hi,

I had some samples that I extracted RNA from, the first time everything worked fine. However, every subsequent RNA extraction, which involved cutting or grinding the same samples on dry ice or in L. nitrogen (so some thawing probably) resulted in low concentrations on nanodrop. I then tried RNA extraction on new samples for the first time, and the concentration readings on nanodrop were high again. I was trying to figure out why the RNA extractions did not work after the first time, and I was wondering if multiple Freeze/Thaw cycles of RNA samples can result in lower nano drop concentration levels. I know that thawing can degrade RNA, but does it degrade it enough that nanodrop no longer picks it up? Thanks.

David



#2 Trof

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Posted 14 February 2014 - 10:19 AM

Nanodrop can't tell you if the RNA is degraded. RNA oligonucleotides breaked-down from the original full-length RNA will give the same reads as intact RNA.

 

But your RNA isolation protocol probably doesn't pick up smaller (partially degraded) RNA or the RNA in the sample is just degraded to pieces.

 

Freeze/thaw cycles are maybe not so good for RNA alone, but are definitelly very bad for a tissue, if you want some RNA from it in the future. It disrupts the cells and degrading enzymes are free to feed on nucleic acids. 

 

My guess is, you just lost most of your RNA after first thawing. If you want to isolate one sample for multiple times, you should cut it into pieces before freezing or store it in non-freezed protection solution like RNAlater, that can be sliced.


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