Total RNA storage in guanidium thiocyanate?

Use this category for the exchange of ideas, methodologies and references regarding the isolation, manipulation and analysis of RNA. (Extraction protocols, Northern Blot analysis, RNase Protection, Differential Display, In Vitro Transcription, etc.)

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Total RNA storage in guanidium thiocyanate?

Postby clutz » Aug 28 2006 10:22 am

Hi!
Does anyone know for how long you can store cell lysates in guanidium thiocyanate (solution D from Chomczynski method) at -80 or -20ºC. Has anyone tried???
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Postby chason » Aug 28 2006 1:02 pm

I don't know about Solution D specifically, but samples homogenized in Trizol and similar reagents (containing guanidium thiocyanate and phenol) can be stored at least a month at -80C
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Postby mchlbrmn » Aug 28 2006 1:08 pm

I've stored samples in GSCN at least for weeks at -20 (and probably longer).
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Postby clutz » Aug 28 2006 1:15 pm

Thanks to both! Is the RNAlater product based on a GSCN solution?
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Postby John Buckels » Aug 29 2006 3:35 am

RNAlater is based on the principle of salting out. The high conc salt in the solution enters the cells and physically precipitates the intracellular proteins, physically protecting nucleic acid from enzymatic digestion

Guanidine and other chaotropic salts simply lyse the cells and denature all protein, so it's a different method of action
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Postby clutz » Aug 29 2006 6:48 am

Thanks, John! I really hope GSCN does´nt damage the RNA. There are some PBL samples from 6 months ago in solution D and our -80 freezer just broke...they were transfered to -20ºC.
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Postby John Buckels » Sep 01 2006 9:05 am

They should be fine, from my experience
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Postby iteo » Sep 08 2006 2:52 am

RNAlater is a mixture of Ammonium Sulphate and Nacl at low (4-5) pH if my ailing memory is correct - I had the formulation somewhere. Under these coditions RNAse is inactive, but can become active upon removal of the salts.


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RNAlater

Postby Culks » Sep 21 2006 9:20 pm

Hi,
On the topic of RNAlater, I was just wondering if any one had the exact formulation? Or know where to find it?
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Postby iteo » Sep 22 2006 2:50 am

I would suggest looking at the USPTO (patents) site. Please note that this is not a recommendation to break patent rights nor to make up the mix yourself.

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Postby John Buckels » Sep 22 2006 6:27 am

to back up what Ian said, tha ambion patent is freely available from delphion, espacenet or any other patent search tool

However personally I wouldn't recommend infringing patent rights for any product. Plus if you buy it from one of the legally licensed suppliers you do get some element of QC and a guarantee the product will work - make it yourself and if you mess up or misread the patent claims or whatever then you've lost your tissue sample and the RNA with it

Plus it's not even expensive. the time you take to make it would cost more than the price of the item!
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RNAlater

Postby Culks » Sep 24 2006 11:09 pm

Hi,
Thanks for the advice, I found the patent but the concentration calculations don't make sense and there are some other ambiguities. I will look into buying it.

Thanks again.
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