siRNA transfection

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montgomj
montgomj's picture
siRNA transfection

I was wondering what methods people found best for transfecting siRNA in vitro. I am working with melanoma and glioma cell lines.

Fraser Moss
Fraser Moss's picture
most people seem to go with

most people seem to go with reagents like

http://www.emdbiosciences.com/html/NVG/transfection03.html

http://www.qbiogene.com/products/transfection/jetsi/jetSI-ENDO.shtml

or other lipids like lipofectamine 2000

here's a protocol

Sometimes between 70-90% or more cells can be transfected with cationic lipids. With a bit of tweaking, and depending on the cell line, you can drive siRNAs into almost 100% of the cells with the following protocol:

Seed cells high, so that they are 85% on the day of transfection:
For one 10 cm dish:
40 ul Lipofectamine 2000
20 ug DNA
100-300 pmoles siRNA

tube 1
add the siRNA and/or DNA to 2 ml SF-DMEM (no drugs added)
tube 2
add the lipid to 2 ml SF-DMEM (no drugs added)

mix these two tubes immediately, for a volume of 4 ml, and then add to cells which have been washed 2X with SF-DMEM. Incubate 3-6 hours, then add normal media. Check for expression or knockdown 36+ hours post-transfection. Knockdown by siRNAs usually last 3-5 cell doublings.

When oligofectamine is used, the identical protocol is used, but:
Seed cells at 15% confluency, perform tfx with 30 ul oligofectamine and siRNAs, then repeat tfx 36 hours later. Analysis is performed when cells are 85-90% confluent.

badcell
badcell's picture
In the cell line I'm using

In the cell line I'm using right now, I've got better results with siPORT Amine from Ambion than from policationic lipid reagents such as siPORT lipid (also from Ambion) or Lipofectamine 2000, which I tested side by side. About 80% of the cells were transfected with the amine reagent, but only 20-30% with the lipidic reagents (Here's a link to the protocol I followed). For other people in my lab,working with other cell lines, Lipofectamine 2000 gave the better results. So I guess it really depends on the cell line used. It's always advisable to test several reagents when beginning work with a new cell line.
Hope that helps.

CSHL
CSHL's picture
We have trouble with Ambion's

We have trouble with Ambion's siPORT neoFX for lymphocyte suspension. the transfection obvious is not working. Ambion is still optimizing this reagent but they just can't wait to make money....

We later changed to Gencarrier-1 for co-transfection of siRNA and a GPCR expression plasmid and got very consistent efficiency (~60%) as well as knockdown effect (>80%).

norman
norman's picture
we have done extensive

we have done extensive testing of several (10+) reagents for siRNA transfection in my previous lab. I hope this information will be useful to someone.
We have tested all transfection reagents with these cell lines: PC-3, HeLa, HT-1080, Mcf-7, C6, Huvec, A549, LNCaP, and Jurkat cells.

So, we had no success with any reagent for Jurkats and HT-1080 cells.
Almost all reagents worked ok with HeLa cells .

Lipofectamine worked for Mcf-7 and ok for PC-3(invitrogen.com) [cytotoxicity was pretty high!!!!]

Gencarrier-1/2 worked well for Huvec, C6, Mcf-7 cells (epochbiolabs.com) [lowest toxicity and price]

Altogen reagents worked best for A549 and LNCaP cells (altogen.com) [but you have to buy one reagent for each individual cell line ) ]

RNAicarrier worked best for C6 cells (epochbiolabs.com)

good luck

Jason King
Jason King's picture
Norman - What levels of

Norman - What levels of transfection did you get with the Gencarrier reagents in HUVECs?

Thanks

samm
samm's picture
Have you tried DharmaCon

Have you tried DharmaCon reagents - i remember having seen some work on melanoma cells using one of their siRNA rgts. they also have some kind of trial pack with multiple rgts to help optimize for your own application. I have howevr not used any of their rgts personally, but they were very successfully used by neighbouring labs.

Jason King
Jason King's picture
I had a look at Dharmaon's

I had a look at Dharmaon's reagents since we ordered the siRNA from them. As we work with endothelial cells, I was interested to see that Dharmacon have tested their DharmaFECT1-4 reagents on HUVECs. Interestingly, whereas their reagents knock down expression of GAPDH by 70%, 45%, 38% and 50%, the "Lipid L" that they use as a (negative) comparison appears to knock down expression to 0% with all 4 reagents. Unfortunately they don't comment on the level of Lipid L toxicity!

I've ordered test packs of DharmaFECT1-4 (Dharmacon), siIMPORTER (Upstate), TransPass R2 (NE Biolabs) and GenCarrier 1/2/RNAiCarrier (Epoch Biolabs).

After receiving the following email from Epoch Biolabs, I'm expecting great things of the GenCarrier reagents...

_________________________________________

Thanks for your interest in our product.
To our knowledge our GenCarrier is much better than those from Dharmacon and Upstate. We also provide deep discount for foreign researchs since we don't have oversea distributor so far. We believe our GenCarrier is the most cost effective and efficient transfromation reagent available on the market. Testing kits are available (0.2 ml @ 1 mg/ml) but we do need to charge you the international shipping which is around ~50.00 by Fedex overnight express.

____________________________________________

Schiffelers
Schiffelers's picture
Electroporation is also a

Electroporation is also a relatively easy way to get good transfection efficiencies. Protocols for DNA usually work fine.