How is it possible that RT-qPCR demonstrate a down-regulation of my interested gene (from 22 to 27 ct) while WB is showing the protein expressed with the same intensity as in cells where the gene is expressed?
Is it possible that cells have developed an ''alternative'' translation mechanism bursting the expression of the protein independently from the level of the transcriptome?
Ps. I am confident with the specificity of my Ab since it was tested in positive control.
Any suggestion? Any example in the literature?
As mRNA is eventually translated into protein, it is usually assumed that there is some sort of correlation between the levels of mRNA and protein. But, protein expression not necessarily be proportional to mRNA levels as the proteins may differ substantially in their in vivo half lives. Proteins can be long lived because of enhanced protein stability due to post-translational modifications like phosphorylation, acetylatylation, glycosylation etc, while the mRNA turnover is quick.
Sometimes, you may also see the other way round where the protein degradation is much quicker while the mRNA levels continue to maintain, especially in induced conditions (for ex., exposure to cigarette smoke).
Most importantly, we shouldn't forget that there is a significant amount of error and noise in both protein and mRNA experiments that limit our ability to get a clear picture.
Worth reading Greenbaum et al 2003 review in Genome Biology. PMID: 12952525
I would suggest you to repeat the qPCR experiment once again and do the relative quantification and determine the fold change of the ecpression level. Since minute changes are often detected u
In qpcr while in protein level, it might not be so much prominent
By considering all technical procedure is ok, If you were comparing patients and healthy subjects and all those patients are related to the specific stage of progression, it can be hypothesized that a number of compensatory mechanisms have been activated to reduce the expression level of the desired gene. and this process takes time to immediately see a decrease in the level of protein expression. And also for cell I think it was the same if you can culture them for longer under those treatment you will be able to see also protein depletion.
Hi Blerta Stringa
A gene does not express under the influence of only one factor or regulator. It is highly regulated by multiple factors/regulators. In your experimental conditions, if you used any general component (not specific) that targets your gene to observe the change in particular gene expression and you may find a positive result in qRT-PCR. But, you may not see protein expression change in western blot.
Here, the possibility is other crucial regulators might have enhanced the translation process of that gene into protein.
However, if you have done any kind of silencing/knockout of the particular gene, then that particular step needs to be more precise so gene and protein expression both should get affected.
Regards
Saurabh
Are you using the same sample (Blood or tissue) for proteins and RNA isolation?. If it is the same, then you have to compare the results with healthy or your defined groups because there is a possibility, the downregulated gene has sufficient copy number for protein synthesis. Sometimes a single copy of mRNA is dominantly synthesized into the protein. Therefore, you have to compare in your groups and check the downregulated gene's protein band intensity. To confirm the downregulation of your gene and protein synthesis, you have to be knockdown your gene and check the protein level.
As mRNA is eventually translated into protein, it is usually assumed that there is some sort of correlation between the levels of mRNA and protein. But, protein expression not necessarily be proportional to mRNA levels as the proteins may differ substantially in their in vivo half lives. Proteins can be long lived because of enhanced protein stability due to post-translational modifications like phosphorylation, acetylatylation, glycosylation etc, while the mRNA turnover is quick.
Sometimes, you may also see the other way round where the protein degradation is much quicker while the mRNA levels continue to maintain, especially in induced conditions (for ex., exposure to cigarette smoke).
Most importantly, we shouldn't forget that there is a significant amount of error and noise in both protein and mRNA experiments that limit our ability to get a clear picture.
Worth reading Greenbaum et al 2003 review in Genome Biology. PMID: 12952525
If I understand correctly, you are comparing condition A to condition B and find a down-regulation in qPCR but not Western (in cells?). Do you find an increase in Western Blot or is it simply unchanged between condition A and B?
qPCR is a much more sensitive method compared to Western Blot, which could explain why you don't see a difference with the Western.
Moreover, as Vinod Nadella pointed out protein levels do not always correlate with the transcript levels. In fact only 40% of variation in protein levels can be explained by mRNA abundance (another worthwhile read Vogel, C.et al. Nat Rev Genet (2012). https://doi.org/10.1038/nrg3185).
Blerta Stringa
As I agrre with previous answers, I would like to state that Protein expression not necessarily be proportional to mRNA levels because the proteins may differ substantially in their in vivo half lives. Proteins can be long lived because of increased protein stability due to post-translational modifications For instance, the protein's half life could be increases due to a reduced rate of degradation. Another possibility is that its mRNA is more preferentially translated during the condition you analyzed. You can check the work by Ronald C. Wek and Alan G. Hinnebusch, who characterized the preferential translation of certain mRNA during cellular stresses.
From a theoretical point of view, it is possible a compensatory mechanism whereby cells try to increase translation even though transcripts' amount is low.
Anyway, you could try to run via electrophoresis products of Real-Time PCR ( both your gene and normalizer) to check the signal got. This could also be useful to understand if you see transcript of your gene of interest.
Frist of all I want to tell you that protein expression is more trusted and reliable than mRNA level...mRNA may be degraded during the extraction and purification procedures..also, protein may perform feedback inhibitions of the target Gene....
More than one protein can be made by a single mRNA. And not always correlate to protein. I would rely on the protein data for expression studies. Try knocking the gene.. With hypomorphic expression of RNA it's difficult to conclude gene silencing at protein level. Give more information on where the protein is localised and what methods were used for silencing. And when were the samples collected after transfection.. The kinetics says in 12 hours u will find the RNA silenced and it will take 24 hours for the protein deplete.. I would go for long term silencing or continuous silencing using sirna or shrna as u wish
Proteins can persist years after transcription has stopped. Just some examples to get an impression on the range of protein stability.
Name Half-Life
Collagen 117 years
Eye lens crystallin >70 years
RFC 19 hours
RPS8 3 hours
Ornithine decarboxylase 11 minutes
(data from the Wikipedia article on 'protein turnover'
Ernst W Müllner thank you for your reply. Any experience with TGFa? It would be very useful...
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