transcriptional activity and mRNA stability

Jimbo Jones jmttsf at aol.com
Wed Apr 24 09:52:42 EST 1996


In article <Pine.SUN.3.91.960424122159.20729B-100000 at dsets.ulst.ac.uk>, 
edq424 at dsets.ulster.ac.uk says...
>
>Dear All,
>
>We are trying to examine gene expression after radiation. We are using 
>Northern Blotting to look at induced mRNA levels. This would give 
>indication of transcriptional regulation of a certain species of mRNA. We 
>heard that it is necessary to look at mRNA stability for that specific 
>gene to see if there is induced expression due to increased mRNA levels 
>or slow mRNA degradation.
>
>Does anybody have any idea of a protocol as how to do this (mRNA 
>stability)?? 
>
>I appreciate your response and ideas. 
>
>
>Kind regards,
>
>Osama
>
>.........................................................................
>Osama A. Al-Assar                  .     ||\       * *           *.
>N. Ireland, UK                     .     ||'\       * *         *.
>EDQ424 at dsets.ulst.ac.uk            .    / |''\       * *       *.
>Research Student                   .    \ |''/        * *     *.
>                                   .    / |'/          * *  *.
>                                   .   /..|/            * * .
>..........................................................................  
                                         
>You need to do nuclear run-on analysis experiments.
i just posted a letter on the news asking for protocols as i am 
having trouble with the one I am using.
Nuclear runons measure directly the transcriptional activity of the gene.  
the idea is that you isolate the nuclei from the cells (or tissue)
 under conditions that preserve the transcriptional machinery (RNA POL)
on the DNA that it was in the process of transcribing.  After the nuclei are 
isolated, then you add back salts, NTPs,, etc. that will allow transcription 
to continue- You add HOT UTP as a tracer- ie the RNAs that are completed will 
be radiolabeled.  Elongation and termination are supposed to continue, but 
INITIATION is not- so you are essentially getting a "snapshot" of the 
transcriptional activity of the cell at the time of isolation.  The rna is 
then isolated and hybridized to your gene of interest- a slot blot of various
DNAs works best- you must include a positive control - a gene you know is 
constitutively active, such as tubulin or actin as a normalization factor.  
You must also include a negative control- a gene you know is not expressed to 
guard against non-specific hybridization.  you then quantitate the amount of 
RNA by densitometer scan, scintillation count of the slot,  or both.
The ratio of the signal generated by your RNA of interest vs. your 
constituively expressed RNA is then used as a measure of the activity.
I am going to reactivate these experiments starting next week-
If you have questions, or come across some protocols- please let me know- I 
have two that I would be more than willing to share with you-
my Email address is 
IO10224 at maine.Maine.edu
That is the letters "I" and"O" and the numbers 10224.

Deena Barry
pHD student-University of Maine




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