>alex at alfred.med.monash.edu.au (Alexander Tzanidis) writes:
> The problem I am faced with is how to isolate total RNA from isolated rat
> As a standard protocol, we routinely use the Cesium Chloride gradient
> technique to isolate total RNA from whole rat kidneys. However, when
> applying the same technique to isolated glomeruli (isolated by seiving;
> approx. 1000 - 5000 gloms/rat), I am unsuccessful.
> The glomeruli, once isolated, are resuspended in 1ml of 5M GTC (without 2-
> ME) and stored at -80oC.
> Could the Chromczynski method (acidified phenol) be applied even though I
> have used 5M GTC rather than 4M GTC/SDS/2-ME?
>> Thank you
>> Department of Medicine
> Monash Medical School
> Alfred Hospital
> Melbourne, Australia
>>>>>Resuspend your RNA (now stored in 5M GTC) in an equal volume of the standard 4M GTC/SDS/2-ME, and then continue on
with the acid phenol extraction. If your RNA is intact before you try this, then you should be able to clean it up nicely. If you are
planning to do this using fresh tissue, perhaps you should modify your 5M GTC extraction buffer. Is there any compelling reason
why you are using it in the first place?? Please let me know. Good luck.