10 replies to this topic

[zienpiggie]

Hi all,

I am just wondering if anyone of you have ever tried corresponding increases in mRNA transcript with changes in protein level? I was using one of those targeted real time PCR assay that basically uses control vs treatment samples and I could screen for the modulation of 84 enzymes at the same time. I went ahead and tried to detect changes in protein expression of some of the genes that were upregulated by at least 15 times or so, but I could barely see changes in the protein expression. And not only for one of them, but for at least four of the proteins I tried. I am therefore wondering, just because the mRNA is upregulated, does it always mean that the protein has to be upregulated as well?

Thank you

[allynspear]

There is actually pretty extensive data out there about how there is no guaranteed correlation between mRNA level and protein level. This is why so many people are doing proteomics now, rather than microarrays and transcriptomics. The protein (usually) is the effector molecule, not the RNA. There are plenty of times where transcript levels don't change but protein levels double or stay the same or go down, which is why no matter what, all microarray or transcriptomics approaches should always be verified at the protein level. I am not saying that microarrays are usless, because transcript levels can be significant and it is important to examine what regulates mRNA transcription because there can be a correlation. In the short term, however, if the cell needs to respond to some stimulus quickly, it is faster to upregulate translation on an existing message, than to synthesize new message. In the long term, to create sustainable increases in protein levels, more mRNA is often the way to go. There are a bunch of papers out there that examine the relationship between global transcript levels and protein levels. I have included a link to a figure from one paper where they looked at mRNA and protein quantitations in yeast:

http://www.ncbi.nlm....3965/figure/F5/

The bottom line is that if RNA and protein levels were directly correlated there should be a well fit straight line through all data points, but the points are all over the place, which is why you always need to be careful how far you take your conclusions from mRNA levels alone.

Best of Luck.

Edited by allynspear, 26 October 2011 - 05:59 AM.

[zienpiggie]

Hi allynspear, thank you very much for your response. I did notice a couple of papers suggesting that there are poor correlation between mRNA abundance and protein expression, but they usually would do the correlation in a particular sample, instead of correlating changes in mRNA and changes in protein content between control and treatment group. I agree with what you are saying that it seems to make more sense to look into changes in protein instead of mRNA since they are the ones doing the work. I can only conclude then from my finding that although the mRNA was upregulated significantly, it did not contribute much to changes in protein expression (lost in translation).

Thank you again!

[allynspear]

I understand the fundamental question: Does a 2 fold increase in mRNA levels lead to a 2 fold increase in protein levels?
This, and all the variations of this question, have been debated A LOT in the battle of the -omics (genomics vs transcriptomics vs proteomics) and the real answer is:

We don't know

I have been trying to find one specific paper, but I can't seem to find it now, where they actually showed that in cells growing under two different growth conditions, cells responded differently to treatment with some drug. In both cases the mRNA levels went up 4-5 fold, but in one media, the protein levels stayed almost the same, and in the other media, the protein levels went up 8-9 fold. I think they showed that this was due to the presence of some microRNA that was continuously repressing translation in one case but not the other. The bottom line is that mRNA levels are relevant, but they are not the only thing controlling expression. Just like in signalling cascades, just because the receptor is activated, doesn't mean that other things downstream can't be blocked.

Good luck with your project!

[zienpiggie]

Hey thank you so much! I will try and look for that paper. If you happen to find it, would you mind sending me a copy?

Thank you again.

[ReResearcher]

I havent found that mRNA changes by much in vivo, nor does protein follow if it does, in my experience. This may be for the reasons given, or because methods to detect changes in protein levels (Western Blot) or only semi quantitative and subject to antibody detection which is not universal, background noise on the blot etc...so it could be that protein does change.

The only time I see HUGE differences is during major inflammatory or apoptotic stress. Homeostatic pathway proteins and mRNA levels are down regulated and stress response mediators, both protein and RNA go up.

[lauxi]

Hi!
I have the same problem, so I'm really interested in this topic!!!
I have two questions:
1) How can I be sure that the proteins remain unchanged and it is not an artefact? How do you discarded it?
2) allynspear: did you find the paper you cited? can you send it to me?

Thanks a lot!

[Sotirios]

Increased levels of protein level in contrast with mRNA levels may be caused due to mRNA half-life

[Tabaluga]

Exactly, I think that mRNA and protein levels cannot be directly correlated because of different half-lifes. For instance, if conditions have changed, certain mRNAs may already be downregulated while the corresponding protein is still expressed and the effect will only be seen later when the existing proteins are degraded and are not or less replaced by new ones.

[Tabaluga]

I've got a question too:

Would it be a good idea, if you want to measure both mRNA and protein expression upon a certain stimulus, to first perform qPCR for mRNA-Levels and have a look at the protein expression at a later time point (depending on the respective half-life of the protein), instead of measuring both at the same time point ?

[lauxi]

Hi Enthusiast!!
Thanks a lot for your comments!
Regarding the half-life, I can´t measure at a different time, because I work with human placenta obtained at birth!!! I know that i have only a "photograph" of this time...
Also, I would expect that different mRNA-proteins have different half-life, but I've these results in at least 4 different mRNA-proteins...

Edited by lauxi, 14 March 2012 - 05:41 AM.