Hello Sehrish;

Firstly; the target protein knockdown and cell viability was assessed at 72 hours post-transfection, not 24 hours. 24 hours is not enough time for the suppression of the target gene.

On the other hand siRNA-pools are better than single siRNAs. But to create a pool is quite a complex. These pools are usually designed to completely cover the target gene sequence. So, to mix 4 different siRNA sequences does not mean you've a effective pool. Thus; I do not recommend doing this. For more information about the siRNA pools; I recommend reading the following article.

I will follow your question.

Best regards.

Quite a few siRNA unfortunately work at the max efficiency that you seeing(40-50%) , lucky one will yield 70-80% knockdown(KD) at max. Having said that, it might not always be a good practice to pool siRNA since that might also pool their offtarget effects. If you have a siRNA that shows a solid 80% down, stick to that. Few of the siRNA might just show a 20% KD, so there is no point in pooling them with a 80% one , you don't gain any substantial increase in KD efficiency  but possibly increase the off target effects.

A few experiments works better using electroporation vs transfection. Can try that.

Avoid serum(use low serum ) during transfection which might reduce KD efficiency.

If your cells are adherent, KD works better performing a reverse transfection(trypsin cells and over lay on KD siRNA mix) so that your reagents will have more access point s to the cells in suspension as compared to adherent ones.

Best wishes

Hello Sehrish;

Firstly; the target protein knockdown and cell viability was assessed at 72 hours post-transfection, not 24 hours. 24 hours is not enough time for the suppression of the target gene.

On the other hand siRNA-pools are better than single siRNAs. But to create a pool is quite a complex. These pools are usually designed to completely cover the target gene sequence. So, to mix 4 different siRNA sequences does not mean you've a effective pool. Thus; I do not recommend doing this. For more information about the siRNA pools; I recommend reading the following article.

I will follow your question.

Best regards.

Hello Sehrish,

i will try to help you. I'm a PhD student so i don't have too much experience  but during this first year,of my phd,  i've done more than one time this experiment. In my opinion you should use a culture medium serum free during the mix of trasfection preparation. I don't know what kind of the transfection reagent you usually use but in my protocol I prepare the mix with the transfection reagent and siRNA in a medium serum free.

Furthermore i use  siRNA-pools, with three different siRNA (i've buy its togheter) and my KD is good and about 60-70%.

in the end you should reduce the incubation and collect the cell afther 48/72 hours after the trasfection.

Good luck!

Best regards

If your transfection efficiency is good and still you do not see reduction in the interested protein  that means the sequence you are using is not effective,  You may have to design other sequences for this expt.  Pooled siRNA sequences may not help if you have already tested each one of them separately.  However, you can purchase siRNA pool from company that might work. 

@Muthusamy

Hello, 

I'm sorry I didn't get "if your transfection efficiency is good and still you do not see reduction in the interested protein, that means...". I'm interested to know how we can know if the transfection is efficient (the efficiency of just the transfection; just the delivery) separately from evaluating how good the sequence of this siRNA is for the KD? Because i'm trying to troubleshoot my KD (almost no reduction) and was wondering if i can know if its the lipofectamine that's not good (its 2000, and many claim they had lots of trouble with it and once they switched to other things like lipofectamine rnaimax or 3000 they got very good results on the first try, or is it my designed single oligo

Would greatly appreciate your input

We use Lipofectamine 3000.  If you are not sure about your cell line transfection efficiency you can co-transfect with GFP or RFP plasmid and check under the microscope after transfection.

Dear Sehrish, pooling siPOOLs is in fact a very GOOD idea as Onur has pointed out and I´m happy he suggested reading the paper. With regard to the off-target discussion I would like to emphasize the point that this paper and many more experiments done in various labs have convincingly shown that by pooling you are not increasing but diluting the off-target effects. We have had a closer look at the smart pools and what we see, that the off target silencing can be reduced – however you need way more complex pools to actually overcome off target silencing entirely. Moreover we have seen that complex pools of siRNAs targeting the same gene make KD more efficient and robust. I am collaborating with a company that generates customized siRNA pools – called siPOOLs.  SiPOOLs in fact consist of 30 individual siRNAs, carefully selected sequences using a proprietary set of design rules and our own work and work of many other labs have clearly demonstrated that both with respect to silencing potency and specificity for the designated target gene siPOOLs are very reliable. If you have trouble to knock down your gene then visiting the web site of siTOOL Biotech for more info isn´t a bad idea, I think. If you want to move on with your pool of four siRNAs then this may well be better than the individual ones. The concentration of the pool should be kept the same. … and increasing the concentration isn´t a good idea if you already work at 25nM –but waiting for a few days later as most of the folks here have suggested most probably is. Onur, thanks for quoting my paper

Good luck with your project

Stefan