siRNA delivery in non-dividing cells - (Jul/10/2006 )

Hello all!

I'm totally new to RNAi technique and I'll have to deliver siRNA in human primary non-dividing cells.

First question is: which is the best company to buy RNAi reagents: Dharmacon, Qiagen, Invitrogen, Ambion?
Second question is: why is it important to have dividing cells when using chemical reagents? I think it is not important that siRNAs enter the nucleus to inactivate mRNAs but I might be wrong!

I have the option of electroporation (nucleofector) or direct injection but I believe these techniques are more difficult to perform.

Thanks for any suggestion.
Cris

Cris,

There is a good discussion on the options of transfection reagents. See pined topics. You have to decide which one is the best for you. There is difference in cytotoxicity among these reagents though.

Cell confluence has something to do with the rate of cellular uptake of transfection complexes. Cells dont divide as much when they are cruded.

Nuclear entry is a major limitation for large saized plasmid DNA which can not easily enter nuclei, esp. when they are (partially ) complexed with transfection agant. Antisense oligos and siRNA should readily enter cytosol and nuclei after transfection. siRNA can work both in cytosol as well as in nuclei, as evidenced in efficent silencing of nuclear RNA.

I would recommend that you consider using a lentiviral/adenoviral shRNA delivery system, as non-dividing primary cells have a very high likelihood of being refractory to lipid-based transfection. Invitrogen has both, and other companies sell vectors that you'd need.

We r using Lentivirus to deliver shRNA to differentiated neurons. My old lab used AAV for the same but into hippocampal neurons and even invivo. Viral vectors r quite effective in delivering the shRNA.

Invitrogen has a kit for Lentivirus. I think stratagene has one for AAV.

Thank you all for your comments

As a first attempt, we will try NeoFx from Ambion and see how it goes. If we're not successful, we'll go for electroporation, viral delivery or even microinjection!

Other probably basic questions are:

- is it essencial to use OptiMem I for transfection or can we use our medium (since it is not serum)?

- is it necessary to plate cells for transfection or can we do it with cell suspensions? What I've read is that it is less efficient with suspensions and I'll expect it will be even more difficult with non-dividing cells.

- is it necessary that siRNAs enter the nucleus to inactivate mRNA? I would think it is enough that they enter the cytoplasm because that's where they mainly will operate, am I right?

Thanks for all your help.

Cris

You can use RPMI1640 without serum for 4 hrs. OptiMEN does not contain serum. Its kind of expensive, but one bottle lasts for long time.

As your first try, you may want to do transfection on plated cells, rather than cells in suspension. Be sure not to seed too much or too little. 70%-80% confluence appears to be good range.

I dont think siRNA needs to enter nuclei to perform silencing.

Thanks genehunter.

I have another basic question (I'm going crazy even before starting the technique!): should I use validated siRNAs, do they really work better than non-validated?
I don't have much time to spend optimizing things (I have to finish writing PhD thesis until the end of the year), so I'm thinking that I should go on validated ones. The cells will be hard to transfect and I need that siRNAs really work.

Grateful for any suggestions.

Cris

Yeah, they tested it before you and it should give you at least 75-85% knockdown, depending which company you purchase from. I like IDT 27mer siRNA.

Better try validated siRNA as it has been tried and tested. I know labs where they have tested more than 11 siRNA to get a sequence worth using.