mRNA level and protein level correlation?
Michael L. Sullivan
mlsulliv at facstaff.wisc.edu
Thu Mar 18 12:26:04 EST 2004
Besides the biological possibilities (like multiple levels of
post-transcriptional regulation that has already been mentioned),
there is the technical issue of how good your measurements are. I'd
be inclined to believe the rtPCR results, but depending on just how
you did the experiment, your western blot numbers my be suspect. I
think it is very hard to get quantitative data from a western both
because of the high degree of variation one gets within a single
blot, and also because western signals are very, very non-linear. If
you are making your measurements by densitometry of film rather than
more directly by using a lumi-imager, that throws yet another
variable into the mix. (I have a western blot right in front of me
right now where a 3 fold difference in loading resulted in only a 50%
increase in signal on the western as measured on a lumi-imager under
conditions where the signal was not saturated!). Minimally, I think
one needs to run a dilution of each sample, and probably run multiple
blots to get believable numbers. If you've done a rigorous job in
setting up your quantitative western (I'm tempted to ask if there
really is such a thing!) and can truly feel confident in that data,
well then the answer may well be biology.
>In order to compare two systems (treated vs untreated) I quantitated
>the mRNA level of my target gene using real-time PCR and quantitated
>the protein level using quantitative Western blotting.
>According to the real-time RT-PCR results the mRNA level is around
>3-fold higher in the treated system compared to the untreated system.
>According to the quantitative Western blotting results the protein
>level is only around 1.6-fold higher in the treated system compared to
>the untreated system.
>I would rather have expected to get the same or even higher fold
>change in protein level. The incubation time after the treatment was
>24 h. The target protein is a transcription factor and I would expect
>such a protein to be synthesized relatively quickly.
>Does anyone have any idea why there is such a discrepancy between the
>changes in mRNA level and protein level (i.e. considerably lower
>changes in protein level than in mRNA level)?
Michael L. Sullivan, Ph.D
Research Plant Molecular
U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706
(608) 264-5397 Phone
(608) 264-5147 Fax
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