Question about doing RNAi in primary cells - (Jan/23/2006 )
Hi All,
Please help me answer this question. No one has done si RNA in our lab before and my supervisor wants me to just get results. [I hate working in industry :-(]. I have NO IDEA from where to even start! I read the instructions on Tusclh lab website but there is nothing specific about "Primary cell culture" and why is it difficult to transfect in Primary cells?? Even before I ask my supervisor to buy Amaxa nucleofector, I have to do my homework and give her a reason for buying it as I believe it is very expensive. So what startegy to use......may be exhaust all chemical ways of transfecting siRNA BEFORE I ask her to buy nulceofector from Amaxa???? Never ever done siRNA transfection before! Please answer my questions below. Thanks.
1- How do I begin? Electroporation or Chemical Transfection?
2- I followed from another post that there are 100s of chemical transfection agents being used......but what about electroporation? Is The "Nucleofector by Amaxa" the only electropotration device available out there?
3- I am working with Primary cell culture and not cell line.....Is it true that Primary Cells are resistant to siRNA knockdown??
4-If so why are Primary cells refractory to siRNA knockdown? And then what strategy can I use to treansfect siRNA into my Primary fibroblast cells?
5- Luckily pre-made si RNA to my gene of interest is available!!! What is the diffrence between using this pre made siRNA pieces and Vector based devilery? Which is better ?
Many Many Many thanks!!
TMQ
Hi,
Have you ever transfected these cells with anything before in your lab? If so, what transfection efficiency do you have? Depending on your method, most transfection reagents will work for vector based siRNA transfections so if you know something works well, that may be the way to go.
Many people use electroporation for primary cultures b/c it improves transfection efficiency, unless you are doing say sorting or microscope work where you can single out transfected cells, transfection efficiency must be very high for good siRNA or RNAi results in fact the pre-made siRNAs are sometimes guaranteed, but only over a certain transfection efficiency... This is something else to consider if you decide to buy the guaranteed-pre-made sequences, they require you to demonstrate transfection efficiency in a parallel control in order to get the guarantee... See the websites, they will sell you something like a flourescent siRNA to use for this control...
Dont really know anything about amaxa nucleofactor...
As far as I know the only problem with siRNA in primary cells is that it is difficult to get a high enough transfection efficiency, not specific problem with siRNA (never tried primaries so don't know this for sure..)
which way is better is also hard to say, depends alot on your system etc...
hope this helps and good luck...
Thanks!
Actually I dont know any other tranfection agent that works as no one in our group has ever tried any siRNA knockdown before.
But it is great that you told me that with pre-made siRNA there are controls available, so that i can know if my experiment worked.
If there is nayon else who has great experience on siRNA transfection in Primary Cells, then please share your experience with me.
PS: Did you mean that siRNA transfectio is difficult in Primary Cells or that any DNA/Plasmid transfection is difficult in Promary cells?
If so: why is that?Why are primary cells difficult to transfect?
Actually I dont know any other tranfection agent that works as no one in our group has ever tried any siRNA knockdown before.
But it is great that you told me that with pre-made siRNA there are controls available, so that i can know if my experiment worked.
If there is nayon else who has great experience on siRNA transfection in Primary Cells, then please share your experience with me.
PS: Did you mean that siRNA transfectio is difficult in Primary Cells or that any DNA/Plasmid transfection is difficult in Promary cells?
If so: why is that?Why are primary cells difficult to transfect?
Maybe I can help here.
Amaxa nucleofector has proven pretty good results finally for us at least in human primary monocytes.
Our problem was to figure out the promotor of the reporter gene to test our siRNA transfection efficiency is essential. Cell type specificy for promotor type expression plays into this.
But yes, amaxa is expensive (the device and the normal kits too). And please be aware that in general primary cells seem to loose transfected nucleic acids much faster than secondary cell lines do.
The biggest advantage of amaxa is they have specialized kits for several cell types and are constantly doing all the time consuming work to further improve the efficiency and viability of the nucleofector solution. You can probably save money with getting the GenePulser electroporator from Bio-Rad and do the electroporation just in PBS instead of needing to buy each time the Nucleofector Kits from Amaxa. But we haven't tried this so far too and a talk I had with a Bio-Rad representative recently confirmed that it can work well - especially now with the new gene pulser offering rectangular wave pulses too - but you need to find a protocol working with your cell type.
And exactly this can take ages.
So if you have the money, can't find a gene pulser protocoll for your cell type and are in a rush I would go for amaxa. They offer now a 96well shuttle too that decreases costs for the single transfection. If gene pulser is available to you, give it a shot, thats cheap in PBS after all. You could ask for a demonstration with Bio-Rad and Amaxa too with your cells.
Chemical tranfections we haven't tried for the primary cells either as normally the efficiency is far lower with those than with electroporation. We use cationic lipids just for secondary cell lines plasmids/shRNa and siRNA.