Help: RNA preparation: very low yields with spinal cord; pH influence?

kang at msvax.mssm.edu kang at msvax.mssm.edu
Mon Jan 22 12:36:26 EST 1996


I am always using Chomczynski's method for purifying RNA. Recently I purified
RNA from Brain, Liver, Kidney, Heart, Intestine, Spleen, Thymus, Muscle and
Spinal cord of mice (3 month old). I didn't see any problem for yield of prep.
Northern showed very clean band pattern. I was using phenol saturated with
water. I checked the pH by pH paper and it looked around 4.0. I don't know if
there is any difference between spinal cord of rat and mouse ewxcept from the
size.



In article <4dhgsq$9mf at news.service.uci.edu>, qnguyen at parker.bio.uci.edu
(Quoc Thang Nguyen) writes:
>	Dear Netters,
>
>	Please excuse the naive question of an 
>electrophysiologist trying to make RNA. I am using the 
>acid-guanidinium phenol-chloroform method (AGPC, also known 
>as the Chomszinski-Sacchi (spelling?) method) to extract RNA 
>from a variety of tissues. I have no problem with rat brain 
>and always get good yield and good purity. However, I am 
>encountering great dificulties with rat spinal cord. The 
>yield appears to be extremely low. With another protocol, a 
>pure phenol-chloroform method (much more cumbersome and much 
>more time-consuming), the yield improves considerably.
>
>	I have tried adapting the acid-guanidinium 
>phenol-chloroform method to the spinal cord, to no avail. I 
>am left with making wild guesses. The last one is the 
>following:
>	
>	The pure phenol-chloroform method uses a buffer with 
>a pH of 9. At this pH, as far as I know, DNA and RNA are 
>solubilized. The AGPC method, on the other hand, uses an 
>acid buffer. I noticed that, after homogeneization of spinal 
>cord tissue in the AGPC buffer, the pH drops down to 3.5. My 
>question (from a molecular biology neophyte) is:
>
>	Is a pH of 3.5 too low for the RNA to solubilize? Or 
>there is something else preventing the extraction of spinal 
>cord RNA by the acid-guanidinium method?
>
>	Thank you for your answers. You can either reply to 
>this newsgroup, or better, to my e-mail address as well.
>
>	Yours sincerely,
>
>
>
>
>
>Quoc Thang NGUYEN
>Laboratory of Cellular and
>Molecular Neurobiology
>Dept. of Psychobiology
>University of California, Irvine
>Irvine, CA92717 USA
>Ph: (714) 824-4730
>Fx: (714) 824-3522



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