Hi Sutupa,

Let's discuss!

First, the nature of the gene?

Then, the experimental conditions and how normalisation was done.

Then,the reference gene/protein used for normalisation.

If these details are provided we could discuss further.

Gene does not express under the influence of only one factor or regulator. It is highly regulated by multiple factors/regulators. Imagine, if in your experimental conditions, you have targeted one factor/regulator to observe the change in particular gene expression and you found a positive result in qRT-PCR, but not in western blot that means other crucial regulators enhanced the translation process of that gene into protein.

Well, if you have done any kind of silencing/knockout of the gene, then that particular step needs to be more precise so gene and protein expression both should get affected.

You need to provide more details as Bhaskar suggested, then members can give more detailed suggestions.

Sutapa Mahata It's natural, I think. No correlation between mRNA amount and expressed protein amount. And isn't it possible to simply explain the amount of expressed protein by qualitative Western blotting?　 If you want to explain the expression level of a protein, I think it is necessary to quantify the protein absolutely .

Hey Sutapa..

What u got is absolutely practical. And several times even i have observed such differential regulation. However, one possibility is that perhaps at that point in the biological system the mRNA gets translated to protein and this is why u get lower transcript but higher protein expression.

Hi Bhaskar,

Thanks for the reply.

Actually, I am treating breast cancer cells with a drug which is showing these type of results. After the drug treatment, I am getting low STAT3 mRNA expression but in the case of protein expression, Stat3 is high.

For normalization, in the case of RTPCR, the reference gene is GAPDH and in the case of Western Blotting, the reference protein is beta-actin.

Hi Sutapa,

I think that one should use the same candidate mRNA and protein for normalization. Only then your normalization is fit for your experiments and interpretation.

May I know the mode of action of the drug? If it is a regulator of translation or induces RNA degradation? Etc, details.

Dear Sutapa, following link may provide you some insights of this phenomenon, where you will also find my answer covering some important aspects of" mRNA-Protein expression" relationship along with some other good answers as well. Hope it will help you out to understand few concepts (but definitely not all) working behind this complex relation in the cells.

https://www.researchgate.net/post/Can_low_protein_levels_induce_gene_expression

Regards...

See the following discussion already opened on RG with interesting opinions and links :

https://www.researchgate.net/post/Gene_expression_and_protein_levels_How_good_are_the_correlations

Hi,

U should use the same house keeping genes for both mRNA as well as protein. And compare the ratios between target gene to the same house keeping gene. A lot of things give u a drastic difference. Few of them are more sensitive western blot detection methods. These reagents amplify the signal to many folds and that's the reason u need to take ratios to compare ur results. There could be possibilities of single mRNA translating to more than one protein. I also recommend most of the answers here.

In this context, mistakes during performance (methods) are the most likely source of uncertainty regarding the described observation (red. mRNA and inclreased protein expression). First of all, I would propose to check protocols of the used methods (WB, RT-PCR etc.) and to repeat the experiments.

After validation of the method you should a) revise the protocol (if there were mistakes) or look for other reasons leading to differences between mRNA expression ang protein generation (from these mRNAs).

This is practical. Higher or lower mRNA levels doesn't always correlate with higher protein expression.