problems with mrna quantitation

Ken Baker BAKERK at FRIR.AFRC.AC.UK
Tue Jan 26 05:39:00 EST 1993


>Fellow netters:

>I have recently analysed my results during a quantitative rna analysis.
>During my analysis I used rRNA as a standard for the year to compare my
>summer hormone mrna levels.  I am dealing with temperate fish so I was
>expecting the summer hormone rna's : rRNA levels to be high during periods of
>rapid growth and the opposite to be the case during the winter months.

>However, I found just the opposite to take place.  That is to say that the
>ratios were high during the winter and low during the summer.  This is sort
>of an indication to me that in, winter flounder, the fish which I am
>presently studying rRNA varies considerably over the year.  Would anyone
>know of another mRNA which remains constant throughout the year which I
>would be able to use as a standard.
>Also, I was wondering if anyone would know if it would be a good idea to
>add a foreign rna to the G.T.C. extraction mixture to show quantitative rna
>extraction when using different probes.

>thanx Juan

>E-mail  Jgill at kean.mun.ucs.ca

Some time ago, I was involved in a project to quantitate mRNA levels specific
for calbindin in chickens under various regimens of diet and hormone treatment.
We found that the best internal standard to use (in an mRNA dotblot) was oligo
dT to normalise for the total mRNA. One word of warning tho, we started off
(like many others) using an actin-specific probe, but found that actin levels
could vary widely with different treatments.


Ken.
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Dr Ken Baker                              JANET : UK.AC.AFRC.FRIR::BAKERK
Department of Protein Engineering      INTERNET :  BAKERK at FRIR.AFRC.AC.UK
AFRC Institute of Food Research             TEL :        (+44) 734 357139
Reading                                     FAX :        (+44) 734 267917
Berks RG6 2EF
UK                          (AFRC = Agricultural & Food Research Council)
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