I tried siRNA transfection against NAK ATPase gene in breast cancer cells using various reagents but it all failed. I did it with transfection reagents like lipofectamine for RNAiMAX, Stemfect and transmessanger RNA. I transfected with 50nM of siRNA. To confirm knockdown I did western blot and realtime PCR.
For the western blot I used antibody NaK ATPase alpha1 (cell signalling) but I found that the antibody was giving lot of background bands so can't tell for definite. I looked for antibodies from different companies and all of them suggest really high concentration for western and high background.
Realtime PCR SYBR - I got primers ( NaKATPase alpha1 ) and did PCR. There was just 20% knockdown with Stemfect reagent. (was it some artifact or for real)
So now my problem is I don't know from where to trouble shoot, is the transfection working well and I can't confirm by my western or PCR, or is it the transfection not working?
Serum-free medium is not a universal requirement; most often, once the RNAi:lipid carrier complexes form, serum can be introduced without a problem. Pen/Strep should be omitted prior to and during the transfection phase.
You need to try some positive controls in your transfection. These could include a fluorescently tagged RNA molecule (such as Dharmacon's siGLO reagent) and an siRNA that is known to work in your system. Many people target lamin A as a positive control; however any sequence that has been previously validated in your lab in this target cell type is fine.
If you have the resources available to get new siRNA sequences, try some pooled targets from Dharmacon or Santa Cruz. Pooled sequences have a higher chance of success that single sequences so are good for feasibility studies. In the meantime, you should invest a bit of effort troubleshooting your blot to get specific signal. Getting membrane proteins in sufficient quantities and to migrate well on an SDS-PAGE is not always trivial. Initially, you might have an easier time checking for knock-down by immunofluorescence or FACS.
have you tried working with hiperfect in opti-MEM ?? and how about you compare your results to a control siRNA for luciferase for example ??
Have you tried increasing you siRNA concentration ?? maybe it's too little...
Princy- can you also elaborate on what time point(s) after siRNA transfection you are checking for changes in gene expression?
@ patrick and Daniel.... I havent tried hiperfect . I have used lipofectamine for RNA i max, stemfect and transmessenger reagents. I use 50nM siRNA for my transfections.
hmmm I will check Si RNA transfection with known control. If i need to increase how much should i increase.??? I did timepoint for 24 , 48 and 72 hrs...
Princy- I've always used siRNA in the range of 20-25 nM, with good success, so I doubt concentration is limiting. It is possible you'd see a more pronounced effect at 100nM, but off-target effects might also increase at higher siRNA levels. siRNA depends on a number of variables, so you'll need to start troubleshooting to see if this is a delivery problem (poor transfection) or a design problem (siRNA sequence is not good or your Na/K ATPase is quite stable and therefore resistant to knockdown).
U can try using Oligofectamine which works better for siRNA transfection. As suggested by other people try increasing siRNA to 100nM (not beyond that) and generally 48h will be sufficient.. For me siRNA transfection at 4% serum concn works better than serum free medium. One major thing is try a pool of 3 or 4 siRNAs with for better efficiency. If transfection efficiency is still a problem u can try for shRNA mediated silencing which would also help u monitor transfection efficiency since shRNA containing plasmids mostly have a GFP...
For lipid based carriers (RNAiMAX, Oligofectamine, etc), make sure you are working with medium that does NOT include Pen/Strep as these can interfere with transfection.
I am not sure how many cells did you seed while performing transfection. You may need to optimize your cell numbers to be transfected. Another imporatnt factor is to opitmize the ration of transfection reagent and siRNA concentration. It is also important, as mentioned above, to use serum free media for carrying out transfection. You can optimize the transfection efficiency of your cells using a positive control such as GFP. For this, you can try Amaxa Nuleofector Device (instead of lipid-based method) which gives high transfection efficiency for difficult-to-transfect cells.
Serum-free medium is not a universal requirement; most often, once the RNAi:lipid carrier complexes form, serum can be introduced without a problem. Pen/Strep should be omitted prior to and during the transfection phase.
Your time points seem appropriate for RNA kd, but I agree you need to optimize the concentration and cell number. Using a positive control will help you do this for your cell lines. Using reagents from dharmacon we have found that you can reliably get good RNA kd. Have you tried pooled siRNA (typically 3 or 4 siRNA against target gene)- which typically leads to best kd - see Mosse et al. Nature 2008 for methods.
lately, I have been transfecting hard-to-transfect cell line with shRNAs and other plasmids using Magnetofectamine. it's also useful for siRNA transfections. the efficiency rises a lot. after buying Magnetofectamine starting kit which consists of: magnetic plate, CombiMag and Lipofectamine (~700Euros) I can do transfections with other lipofectants instead of Lipofectamine (we use homemade PEI) and only buy CombiMag to decrease costs. with this method one transfection costs around 1 euro and it's very efficient. more you can read here:
http://www.ozbiosciences.com/
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