siRNA transfection of primary neurons

Has anybody managed to successfully transfect primary cultures of
cerebellar granule neurons with short double stranded siRNA? If so,
what transfection reagent did you use and how much RNA and transfection
reagent needs to be combined to achieve optimal transfection? Any
additional details that could be provided would also be greatly
appreciated. Thank you.



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Michael J. O'Hare, Ph.D.

Tel: (613) 993-4294 | Fax: (613) 941-4475 | email: [Only registered users see links. ]



Institute for Biological Sciences

National Research Council of Canada

1200 Montreal Road, Building M-54

Ottawa, Ontario K1A 0R6, Canada

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O'Hare wrote:
We tried this with primary rat neurons/astrocytes/co-cultures, and
nothing would get any nucleotide in. Lipofectin, Lipofectamine 2000,
Genjuice etc. We ended up electroporating, though 50%+ of the cells
die. I'm afraid I can't tell you how this will work for siRNA because
we were using luciferase reporter constructs.
Have you though of making a lentiviral vector for it? Once it's made
you can pretty much bet on it working on every cell type, and in vivo.

In article <1164366802.756109.253140@j72g2000cwa.googlegroups .com>, "DanSolo" <[Only registered users see links. ]> wrote:

People electroporate siRNAs into all kind of cells all the time.
Should be no problem.

DK

Here are some references from Invitrogen cell line database:
Source: Nucleic Acids Res 2001 May15;29(10):2052-8
Comments: SK-N-SH cells plated on glass coverslips were transfected
with different...
Scientific Application(s): Transient Transfection
Product Name and Cat No: Lipofectamine™ 2000 (11668019)
3. Source: Proc Natl Acad Sci U S A 2002 99(23)14716-14721
Comments: SK-N-SH cells were stably transfected with Lipofectamine
2000.
Scientific Application(s): Stable Cell Transfection
Product Name and Cat No: Lipofectamine™ 2000 (11668019)

They also have a protocol but thats for Invitrogen proprietary Stealth
RNAi which will be easier to transfect.

Invitrogen have a new transfection product Lipofectamine™ RNAiMAX
Transfection Reagent designed for siRNA but I have no info for neuronal
cells. Maybe worth giving a try.

Actually there is a protocol for SK-N-SH neuroblastoma cells for
Lipofectamine RNAiMax. Go to the invitrogen link:


[Only registered users see links. ]

StewJW wrote:

Maybe you'll have more luck with Lipofectamine 2000 than we had with
our rat primary neurons. It was indeed very good at getting siRNA into
our established human/rat astrocyte cell lines. I also wouldn't advise
you to use any reagent that is recommended for "Stable Cell
Transfection" only (although I see Lip2000 is recommeded for both), as
you can generate stable lines with very low transfection efficiency.