Hello,

mRNA is well known to be eventually translated into protein. Usually we conceder, there is some correlation between mRNA and protein expression; But, there are many possible reasons for poor correlations as well. First of all, there are many complicated and varied post-transcriptional mechanisms involved in the turning of mRNA into protein which is not yet well-defined in the scientific world! Secondly, proteins might differ substantially in their in vivo half-lives; and/or there is always some significant amount of error and noise in both protein and mRNA experiments which are the limits for us to get a clear idea to draw a conclusion. I think post-transcriptional modifications of mRNA might be one of the reasons for the disconnection between the protein(s) corresponding to the mRNA expression, such as; phosphorylation (e.g AMPk), ubiquitination (e.g PRMT1), methylation, acetylation (e.g SIRT1) or GlcNAcylation.

Kind regards

Debasish.

What's your 'failed to get proper mRNA expression' mean? You failed to measure mRNA levels or the trend of mRNA changes are not consistent with the protein levels?

Have you performed a reference gene stability assessement (i.e. GeNORM) for your RNAi experiments? 

This could be a reason for discrepancy between transcript and protein. Normalization to unstable genes are known to give erroneous results. 

A quick way to gauge this would be, assuming homogenous samples with the same RNA input, your Cts for the  reference gene should be similar.

Dear Han Zhang

'failed to get proper mRNA expression' mean the trend of mRNA changes are not consistent with the protein levels

Dear Can

yes. I have checked the quality of my RNA through gel electrophoresis before starting the qRT-PCR experiment

That's different. What i am asking is how do you know the gene you are normalizing to is stable. If this concept is foreign to you, recommend this site:

http://normalisation.gene-quantification.info/

Dear Can

Yes I did the normalization

Which method did you use?

Hi, Harun.

for accurate quantification in qPCR, it is crucial to design primers allowing  efficient amplification (efficiency should be between 90 and 110%, possibly as close to 100% as possible) to ensure that you have doubling of your target at each PCR cycle. In addition, the choice of a reference gene  that is stable in your experimental conditions is also fundamental (see Can suggestion). Also check if primer pair for your gene of interest recognise the different transcript isoforms that are translated in the polypeptide recognised by your antibody (

This ensures that you are approaching your biological issue correctly from the  "technical" point of view (of course, also for Western blotting you may have to do controls that you are only recognising the protein of interest and normalising properly). Said that, if you still observe a discrepancy between transcript and protein levels, there may be biological reasons for that: for example different synthesis/degradation rates for transcript and protein, stabilisation of protein by physiological or pathological partners, and so on. Therefore, although often transcript and protein levels correlate, this is not a "must"!

Good luck for your experiments

For an in depth discussion of the relationship between the mRNA expression level and the protein level in many cell types you can go to:

 "A gene's mRNA level does not usually predict its protein level"

A review by Nancy Kendrick, Kendrick Laboratories, Inc., 1202 Ann St, Madison, WI 53713

[email protected], www.kendricklabs.com

This review can be accessed by Google Search or by going to the Kendrick Laboratories web site. You will see that the correlation coefficients for the relationships between the two variables( R Squared) on average are near 0.5. The only way you can be certain that there is a linear relationship between mRNA and protein levels is to measure the levels using validated approaches such as mass spec for protein and validated mRNA quantitation (q PCR can work if properly validated). Westerns are much more difficult to quantitate than one might expect (in my experience). 

Good luck   

There are two very different possibilities. First and perhaps most likely, as others have eluded to, measuring the concentration of RNA by qRT-PCR depends on the quality of the RNA prep and on the efficiency of the PCR. There is lots of advice in the literature and the posts above about  how to design good qRT-PCR primers and controls.

Assuming that you have this step sorted, then the other problem is that, in general, mRNA concentrations are indicative of the amount of a given protein being produced at the moment the cell is lysed to prep the mRNA you are going to quantitate, but may not be indicative of the total  amount of that protein in the cell, which also depends on how that protein is used and metabolised subsequently. So for example histone mRNAs are under very tight control: histone mRNA synthesis begins just before S-phase, is co-ordinately regulated in parallel with new DNA synthesis, and the mRNA is rapidly degraded at the end of S-phase, to allow the production of enough new histones to  form chromatin on the new daughter cell DNA. If translation is blocked, the histone mRNA remains stable, but if translation is allowed mRNA degradation occurs so that the half life of the mRNA is measured in minutes. On the other hand histone proteins are present throughout the cell cycle, and although there is some histone turnover, in large parts of the chromatin histone proteins have predicted half lives of months to years in post-mitotic cells.

Although this is the most prominent and one of the most extreme examples, you should consider whether the differences in measurement you see are simply differences between time when your protein is being synthesised rapidly, and measurements of its survival post synthesis.

Thank you all of them for your nice comments. Again I will do qPCR according to your recommendations

Couple of things to consider:

1-How certain are you that your Western blot results correspond to the right product? Did you perform any validation experiment (siRNA, protein positive control etc..)?

2-What kind of qRT-PCR values are you getting for your RNA? Are you picking up a strong (low Cp values; e.g. 22 and lower ) or a weak on (> 26). If your signal is > 26, it will likely (especially if several primer sets give you similar values) mean that your protein will not be detectable by straight Western blot.

3- It is possible to get compensatory responses and that reduced mRNA levels may reflect negative feed back from increased protein activity for instance. This is however the exception rather than the rule.

All things done properly starting from Trizol RNA extraction process, sometimes mRNA expression does not correlate with protein expression due to posttranscriptional modification and this is well known. When this happens, it might open another field of idea for exploration.