Hi Petro,

1) As to your negative control-this shouldn't hapeen but perhaps the scrambled siRNA might have complementary sequences to an essential gene but given that you've tried two different companies... maybe this is as simple as a mislabeled tube? Did you have an experimental group that was surprisingly low?

2) 48 well plate will require you to keep a pretty low volume of media so that the siRNA can get to the (I'm guessing) monolayer of cells. This will cause you to have to change media more often and possibly lift cells but if you are careful and your scaling is precise you shouldn't have a problem.

I've done similar experiments with 96-wells and Lipofectamine 2000 with success. Are you doing WB or ELISAs to confirm your silencing?

Hi Felix,

1) By experimental group meaning the treated cells with my siRNA that targets protein X? If so, then I am seeing expected results with respect to cell death as protein X is essential. My no treatment and silentfect groups are highly similar, using the alamar blue assay, but the neg control siRNA has some weird effect on the cells. I was also thinking that the neg control siRNAs are exhibiting major off target effects. I don't know how to go around this.

I've done qPCR and WB on the protein X, and I observed significant knockdown. The weird thing here is that the Neg control group shows no changes to the protein X. This should rule out that the neg control siRNA was possibly contaminated with the siRNA for protein X. I prepare the cells in a 6 well plate for qPCR and WB analysis, and looking through the microscope, the Neg control group looks fine and comparable to no treatment. Is it possible that it could something with concentration of silentfect per wel?

2) I should have include more specifics: I expose my cells to silentfect and respective siRNAs for a total of 24 hrs. After this I change the media, and assay at appropriate time points. Transferring to the 48 well plate, I was looking forward to adding 200-300 uL of media per well (volume typically used in 48 well plates) after the 24 hr transfection exposure time.

Should I take my chances at using an siRNA that targets a non-essential protein as a negative control? Any thoughts?

"Is it possible that it could something with concentration of silentfect per well?"

-Certainly, if human error delivered more siRNA via silenfect to your negative control then you should expect more off target effects. Also, 50 nM of siRNA via lipofectamine is already pretty a pretty high amount, have you tried lower amounts? perhaps your delivery was too effective and that's why you are observing high off target effects.

"Should I take my chances at using an siRNA that targets a non-essential protein as a negative control? Any thoughts?"

-Since your qPCR and WB showed no knockdown of protein X in your negative control you seem to be on the right track-You could use siRNA for a housekeeping, constituively translated protein to show that the observed, significant cell death is due to silencing of protein X.

Petro,

Have you tried using a control where the Si RNA sequence is similar to your test ie the one against protein X but with 3 or 4 mutations? My personal feeling is that this would be a more specific control. Sorry that this not a direct answer to your question

The other thing you could try is to vary the lipid to Si RNA ratio with both your controls and test siRNA and hit a sweet spot where you get death with test and nine with control. Hope these suggestions are of help

Petro,

You need to consider two issues:

1- siRNA concentration: what form of siRNA are you using? Pool? Individual?

2- siRNA company: Ambion has a good negative siRNA in compare to Dharmacon as cited in one of the siRNA high-throughput optimization papers.

Some recommendations:

If it is the first time you do this experiment then you need to consider the following:

- seeding density

- lipid type

- siRNA type, company, and concentration

- lipid concentration

Hopefully that helps :)

Felix - Thank you. I have actually compared Biorad's silentfect to Lipofectamine RNAiMax, and for some reason the cell lines I am using behave better to the RNAiMax, in regards to relative cell proliferation. What's more is that the cell lines are not affected by the negative control siRNA when I use RNAiMax, whereas the silentfect itself is relatively more toxic, and there's significantly more toxicity using the same negative control siRNA. I'm not really sure why RNAiMax would be that much different than using silentfect, but this was an interesting find. Should have used RNAiMax from the start. It's really worthwhile looking at different transfection agents for siRNA. There are major differences among them. An important lesson I've learnt here.

Krish - My next step in my research is to compare a scrambled sequence. I did run some optimization experiments with the negative control siRNA and transfection agents (RNAiMax and silentfect), and it seems like I hit the sweet spot. Since RNAiMax really lifted the negative control siRNA toxicity, it is not a major concern now.

Fawzi - I'm using individual siRNA, and from what I understand, they should be 27-mers. As with transfection protocol, I saw the reverse transfection with around 10000 cells per well works the best for me. I've also done some optimization experiments.

Thank you all for your helpful responses. I really do appreciate it.

Cheers,

No problems Petro.

Well, I guess you went too far by using a final concentration of 50nM with individual siRNA. We do 50nM with pool of 4 siRNA as each one will end up with 12.5nM so altogether 50nM. But when it comes down to individual siRNA then 10nM should be really enough. If 10nM doesn't work then you may go 15 or 20nM, but I wouldn't recommend higher concentrations with individual. Give it a go with these low concentrations, it should work, hopefully :).

We have used two different unrelated RNA as negative control for our siRNAi, unfortunately we have encountered non-specific effect on our worms.  We have no idea how and why happened.  Any input?

Dear Benwen, Li

I have encountered the same problem with you ,have you found any answers and resolution about the question.