Dear Mamoun Ahram ,

I hope that your siRNA is not working or your working concentration of siRNA is not sufficient enough to suppress the respective gene expression.

Have a look on the following points, hope may be helpful to you.

1. siRNA working concentration will be different from cell type to cell type. Find out which concentration is best.

2. Make sure that your siRNA is working.

3. Generally siRNA is more effective between 48-72hr. Find out which time point is better.

Regards

sarat

Hi! Before trying to give you an explanation I need to know some more about your experiments.

1) Are you using a single siRNA sequence or more then one?

2) Did you design your siRNA sequence or they are commercial or found in the literature?

There is the possibility that your miRNA mimic or siRNA acts by an enhancer by targeting a particular UTR

Thanks. Both the siRNA and miRNA are commercial (Thermo Fisher) and claimed to be validated. Two concentrations were used: 75 and 150 nM. The recomended concentartion is 75 nM. We doubled it to 150 nM. Only a single sequnence (single miRNA and single siRNA) is used. Validation by immunoblotting was done 72 hrs post-transfection.

in case of the siRNA experiments, Although the targets proteins were not suppressed or down-regulated, we got the anticipated results at the morphological level and actin structure level. We just wanted to show that the protein levels were reduced to explain our positive results. As for the miRNA experiments, the results have already been published (the MMP-13 paper in m contribution). I had the explanation and it was accepted by the reviewers.

How do you know if they are working other than immunoblotting of target proteins.?

Hello again!

You can try to check the mRNA level of your target protein by qPCR. If your interfering sequence are working well you should see a decrease in the levels of target mRNAs. However is conventionally required the immunoblotting of your target protein.

Another explanation for the phenomena that you are observing it's the possibility that as a compensatory mechanism the cells increase the stability of your target protein. In this case you should see its reduction later then usual. You could try to increase the timing of your experiment till 96h also or using more then one sequence for eache target gene.

I hope this could be helpfull for you.

I think the increase in the protein concentration between treated and untreated samples could be because of the difference in the number of cells. The siRNA and miRNA can be increasing the proliferation after treatment. If you could collect the cells, count them (e.g. using hemocytometer) and prepare the protein lysates from same number of cells (for all the conditions), it might provide some insight. Protein estimation could be done after the lysis step.

Fluorescence/Confocal imaging (Immunocytochemistry, ICC) can be performed to see the protein expression inside the cell. Alternatively, the cells collected from monolayer can be made into a pellet and embedded in paraffin-wax to make measurements similar to immunohistochemistry .

RT-qPCR can be used to monitor the changes in target mRNA levels. siRNAs are fully complementary to the target mRNA resulting in AGO2-mediated slicing. MicroRNA also destabilizes the target mRNA most of the time (~80% of the time [1]). However, as you might know that there may/maynot be a correlation between mRNA and protein levels.

[1] Eichhorn, S.W. et al. (2014) Mol. Cell 56, 104–115. Article mRNA Destabilization Is the Dominant Effect of Mammalian Mic...

Dear Mamoun Ahram,

How many transcript variants do your proteins have? Is your siRNA is targetting to 5'UTR region or in the mRNA? Which transcript variant is targeted by your siRNA?

Is your antibody detecting all the types of transcript variant?

If your siRNA is targetting 5'UTR region then it can suppress all the transcript variants otherwise please check which transcript variant is targeting.

Regard

sarat