Help! Quantification of total RNA, equal loading

Peter Nilsson peter.nilsson at klinkemi.umu.se
Wed Jun 7 00:50:28 EST 1995


In article <3r32j2$8dp at geraldo.cc.utexas.edu>, soohwan at mail.utexas.edu (Soo-Hwan Kim) says:
>
>I found difficulty in loading same amount of total RNA in wells of 

Hello, my experience is that it is not easy to determine the exact concenration
of RNA in solution. I have been looking for a better way to do it than A260/A280
but havn't found anyhing yet. A problem with A260 is that lots of other substances
potentially found in your RNA prep, such as proteins, phenol, carbohydrates, DEPC 
and so on, absorbe at that wavelenght. You get lots of interference and different "[RNA]"
depending on the purity of the prep. 

The only way I know of to avoid this effect when doing quantitations is to use an internal
control. That means that if you do northerns you hybridize the filter first with a probe
detecting something that is constantly expressed in your samples. Then you strip the filter 
and detect your gene product. Then you can normalize the signals between samples to the control.
The problem is to find good controls. In human systems, which I work with mostly, actin is one
commonly used internal control, GAPDH another. 

HopE I helped you somehow, 

Peter Nilsson
Clinical Chemistry Department 
Umeå University Hospital
Sweden.
 



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