Did you see increase in expression of these genes or increase of translation in protein? I'm asking, because this two things are very differents. One case is increase in gene expression only and no difference in protein, other thing is decrease in gene expression but increase in amount of protein. In the first case, microRNAs can be acting modulating the translation of mRNA, while in the second case the cell can be using alternatives pathways (pathways that don't use the genes that you trying to silencing) to translate that protein.

I hope that helps you.

First I agree with Pedro's answer.

To give you additional possibilities I would like to clarify that you have normalized the amount of protein in each lane to the same amount, for example by testing the expression of a house keeping gene such actin or others?

Thanks to both of you for taking the time to post an answer. 

To Pedro first, I see an increase in the amount of protein, indeed. I haven't checked at the mRNA level yet. I am going to do it.

Ghil, yes I normalized both with GAPDH and B-actin. Very neglectable difference between my samples. 

I agree with you Pedro, but I'm still skeptical as it happens for the three of them, not just one... and I never had this kind of issue before. 

Ok Carole, in such a case that is indeed not something I could easily explain. :-(

Some additional thoughts for you:

• Are the sets of siRNA from the different companies the same designs or different? DO you use them as singles or pools?
• Primary cells are usually hard to transfect, and their efficiency may be very low. Do you have a positive control for the delivery? For example siGLO from Dharmacon, or using siRNA towards PLK1, that is known to induce caspase dependent cell death. If yet what efficiency do you get?
• Did you change the transfection reagent (even a new lot may sometimes cause reduced transfection efficiency)?
• What concentration of siRNA do you use. People use typically 20-50nM in cell lines. Given that you are using primary cells, however, you may need to increase even to 100nM, if the transfection efficiency is low.
• One more good to know - the gold standard for siRNA, is that an siRNA is functional when it is reducing the mRNA by >=70%. This however does not necessarily coincide with the protein as was mentioned above by Pedro.

Hope that this will be helpful for you.

Thank you Ghil! I appreciate your help.

I do not have any positive control for the delivery, this is indeed something I should include in my experiment. I use a 10nM concentration, the reason is because I'm pursuing a study based on results that came out from a high-scale siRNA screen that was using that concentration... 

I'm also going to check mRNA levels.

Thank you again. 

Carole

Carole,

delivery control is very important,try those I suggested above.

It is enough that you target only 50% of the cells to see an increase in the expression rather than KD.

As to the concentration of siRNA it is very much cell line dependent (also passage # of the cell line used), transfection reagent dependent, confluence dependent and so on.... so I would suggest boosting the siRNA to 25nM & 50nM, and maybe try a different transfection reagent.

Ghil, 

Other factor that can affect the transfection efficiency is the time that the cells are exposed to lipofectamine, because its toxicity. I suggest  expose cell to lipofectamine for a maximum of 5 hours. And regarding the concentration depends of the siRNA that will be used. In my case, I transfect adherent cells with a specific anti-miR using a concentration of 75 nM, even in the literature one study using the same cells were transfected with 50 nM, but with other anti-miR. So I suggest that the Carole make a dose curve to determine the optimal concentration.

Other thing that Carole must pay attention is about the confluency of cells at the time of cell exposion to lipofectamine. Is important that the confluency is around 65% until 75%, because the cells need grow after the transfection.

Pedro,

 I agree with you. There are many variables that can affect the results (some you mentioned and some I did , but definitely others can be also important), that is the reason why the inclusion of a positive control is important for Carole.

Best,

G

Thank you to both of you! I'll keep you posted in a couple of days!

So just a quick update. It looks like antibodies did not work, as qPCR results confirmed the knockdown of my genes of interest... 

Antibodies? But did you trying to do a transfection of siRNA or antibody? I discussed it with my colleagues and we agree that is impossible to do a transfection of an antibody, using the lipofectamine protocol. Maybe if you try electroporation, but even that we don't have certain if it is possible. I did see some studies using flow cytometry that marked proteins with antibody inside the cells to see it, but in this case you cannot see gene expression.

Hi Pedro,

I was talking about the antibodies that I used to check the efficiency of my transfection didn't work. Actually the transfection by itself worked, since I had a big decrease of gene expression by qPCR analysis.

By curiosity, If your transfection worked, why do you need check the efficiency of the transfection ? Some Peer Reviewer asked? Or do you need this to improve the transfection for spend less reagents?

I always check the efficiency of my transfection, noone asked :) However, this is something reviewers usually ask, don't you think? Thank you Pedro for your help!

Actually checking the expression of your target gene by qPCR and/or WB is the only way to make sure your transfection worked. Not sure if we misunderstood each other... 

Yes, I understand it. But if you silenced the gene expression, is expected that you haven't protein expression, right? But in your case, did you see protein expression, is it? As I mentioned before, maybe another pathway is capable for translate these proteins, called a non-canonical pathway. How many bands these proteins have in WB? Have these proteins more than one subunit? Are there differences between the protein expression of control and the transfected cells?