For my project, I need to use a plasmid that produces eGFP upon influenza infection. This is done by expressing RNAs containing antisense eGFP flanked by the 5' and 3' UTRs of the influenza NP, resembling a viral gene segment. (eGFP is only produced when there is active influenza polymerase in the cell) I have to knock down this eGFP with eGFP siRNAs cotransfected in with the plasmid using lipofectamine 2000. Based on sequence analysis, they target along the eGFP portion of the transcript and their target sequence is not interrupted by the UTRs.
Two months ago (late September), I was able to get knockdown of eGFP as meausred by flow cytometry, as I was able to do a month before that. At the beginning of this month, I tried the experiment again and this time, no knockdown. The transfection efficiency has not been low by our standards, so it is not likely a transfection problem. The same batch of eGFP siRNA robustly knocked down eGFP produced by a continuously expressing plasmid (pCAGGSeGFP) and I have confirmed in the records that the virus-dependent plasmid produces eGFP and not GFP. (I also tried GFP and eGFP siRNAs in parallel and neither worked.) I tried a new stock of plasmid, and tried the assay using fresh cells. Still no knockdown. I am running out of ideas for solutions to test. Could anyone suggest something that might be wrong here? I apologize for the length. I wanted to show that I have tried all of the basics.
In cotransfection experiments with even the strongest expression plasmids and lip2000 we got very high kd rates. 293t cells amplify plasmids containing sv40 promoter sequences resulting in extremely high expression levels. So maybe you need to increase sirna concentration up to 100nM final. Did you sequence your assay plasmid and you're sure the sequence matches the one from the sirna?
I would look to the simpler explanation first. Shelf life of siRNA shouldnt be a problem unless it's degraded. You can run some on a PAGE gel to take a look at it, or just buy a new synthesis. The $300 for an siRNA is nothing compared to cell culture time and expense. Also, are you sure you are achieving any knockdown? Perhaps a control against an easy to assess gene?
I have not yet screened for mycoplasma. The problem persisted when I tried using a fresh flask. I get a 50-60% reduction in the mean fluorescence intensity of my cells when cotransfecting my continuously expressing eGFP plasmid along with my eGFP siRNAs as compared to scrambled siRNAs, so I am certain that I am getting knockdown with my siRNAs. I am going to see about getting my assay plasmid sequenced. The problem is that it worked two times before it stopped working. Is it possible that those experiments were flukes or noise that could be covering up the real problem?
Back to the SV40-P story, as far as I know, the pCAGG vector should be extraordinarily strong in 293 T! If your assay vector is not as strong (I would guess it's at least one log less strong, as compared in FACS), then you should expect an even stronger KD for the latter as for the pCAGG. If not, there is something wrong and I think it's the sequence. (one central mismatch is enough to abolish any siRNA effect...). Of course, I like also the idea of the Gel (15% native PAGE/TBE or TAE), although siRNA is very stable when stored at -20, even with frequent freeze thaw cycles (s. Eric Lader). Can someone tell me how Mycoplasma should be responsible please?
First make sure if the cell and siRNA are good, then can try
1. Do twice transfection with the siRNA in two continually day
2. Wait for 72 hours after first transfection to check the knockdown
You did all the test for controls after first and the second unsuccesful block, if i read well. Did you still repeat the real experiment to get (non)response? Go ahead, and repeat, and repeat. Mightbe something just goes wrong. A bad batch of tubes, pipettetips, etc. Try to variate the possible factors over.
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