it would help to know what you're stimulating and measuring as "immune response".  in the meantime here is a possibility.  when you use SiRNA, you're likely using something like lipofectin to get it into the cell?  that perturbs the membrane and perhaps stimulates the inflammasome.  so the cell gets at least partially activated.  if you're comparing the SiRNA to an empty vector, then the empty vector is also causing partial activation and the differences between control and experimental will be small.  when you use ShRNA, you wait for much longer to get a stable line, so the membrane perturbation will have settled down and your cells will be more in a "resting" state.

in any case, could you offer some more detail as to what you're doing?

One of the oligoes might be causing an off target effect (a very potential problem with these oligoes). Make sure to double check for off targets and design and test at least three shRNAs. Once you get a consistent phenotype, make a rescue experiment, putting back the cDNA of the gene you are targeting.   

Hi,

Thank you for both of your answer. I would like to give more information about my experiment.

In my system, I knockdown lipid kinase which is PIP5Kalpha, producing lipid at the plasma membrane. Then, I stimulate cell with LPS and measure cytokine production such as IL-6, IL1-beta or IFNb, etc. 

I have used this two methods on BV2 macrophage cell line previously, both worked nicely and consistently, at that time I also try rescue experiment as well. So, I think there are no off-target effect. Because BV2 is unstable so I change to Raw264.7. Basically, everything is the same, only the target cell is different. Therefore, I can not figure out where is the problem. 

I also thought about posibility that Raw263.7 may not like puromycin so I reduced puromycin concentration as much as possible but finally its still the same. 

Do you have any other ideas?

Hi,

1) How much knockdown of your target molecule do you get when you use siRNA  to transfect your RAW264.7 cells?

2) Have you compared your RAW264.7 cells transfected with siRNA, as a control, simultaneously when you take your stable shRNA clone for your experiments?

3) I am confused! What do you mean when you say siRNA decreases immune response? Is it that the siRNA showed decreased levels of cytokines compared to shRNA stable clone or siRNA transfected cells show decreased amounts of cytokines compared to cells transfected with your control scrambled siRNA? This is not clear in your question:

4) Although it is highly unlikely to happen when you electroporate your cell line with siRNA, have you checked the viability of your BV2 or RAW264.7 cells transfected with your siRNA before the experiment and at the end of your experiment? If electroporation with siRNA decreases the cell viability than your control transfected cells then it is of no wonder in observing a decreased production of your cytokines relative to shRNA.

5) If there is no change in cell viability, then I would ask you another question. How much time your cells need to proliferate/multiply/divide? If the proliferation time is shorter than your experiment time, this might explain why you get decreased levels of cytokines in your siRNA approach relative to shRNA stable clone. Because your siRNA effect is only transient and is not stably transferred to daughter cells.

There could be still more details one need to look at, because, God is in the details, as Polly Matzinger always says!

Cheers.

Dear Amarendra Varma,

Thank you for your detail question, here is my answers

1. I knockdown target gene by SiRNA using Lipofectamin2000, and ShRNA using lentivirus system. Both of case the knockdown efficiency is around 70 - 80% in comparison to control SiRNA and Non-target ShRNA, respectively.

 2. I have never compared the expression of target gene on stable knockdown cell with control SiRNA cell, but I compare non-target and stable knockdown cells with wild type cell. Bascially, there is no significant difference between non-target ShRNA cell and wild type cell by means of protein expression and mRNA levels. 

3. in my system, I knockdown the target gene, then stimulate the cells with LPS to activate immune response, finally I measure cytokine production to see whether my target gene have negative or positive effects on immune response. At the basal levels, immune response is equally among the cells, I just saw the difference after stimulating cell with LPS.

4. I did not see the difference among the cell after transfected, therefore I have never check cell viability berfore. 

5. Normally I just stimulate cell up to 6 hours. therefore, cell confluence is still ok and withtin 24 to 30hours. I also check the knockdown after 30 hours already, they show nice knockdown efficiency. 

so, I still dont know exactly now 

 /////////////////////////////// 2. I have never compared the expression of target gene on stable knockdown cell with control SiRNA cell, but I compare non-target and stable knockdown cells with wild type cell. Bascially, there is no significant difference between non-target ShRNA cell and wild type cell by means of protein expression and mRNA levels. ////////////////////////////

Ok, then how can you say that the immune response is decreased in siRNA compared to shRNA, when you did not test the cytokine levels in both siRNA and shRNA cells in one single same experiment? From your question it seems that you have tested siRNA in BV2 cells, and never in RAW264.7; and shRNA in only RAW264.7. Is this true?

If it is true please check siRNA in RAW264.7 cells too along with shRNA. To further confirm that the shRNA is indeed increasing immune response also test your shRNA in BV2 cells too.

If you have already done it and you still see contradictory results then wait until Polly Matzinger comes here or you can verify her answer which she has posted before.