Mortality with Lipofectamin RNAiMax and RPE-1 cells - (Jul/11/2007 )

Hi,

I've made a siRNA interference experiment and all my RPE-1 cells went dying after a night. They were 50-60% confluence, with no antibiotics in the medium. I used Lipofectamine RNAiMax from Invitrogen and even my controls with siRNA directed against Luciférase died.

Does someone have an idea of what could have happened ?

Thanks

-Kooothor-

QUOTE (Kooothor @ Jul 11 2007, 04:20 AM)

Too much lipofectamine can kill cells upon transfection. You need to optimize it. May be you could increase cell confluency or reduce lipofectamine while transfecting.

-scolix-

What is RPE-1 cell? What growth medium does the cell grow in and what medium do you use for preparing the transfection mixture? If the cell grows in serum free medium and you use opti-mem reduced serum medium, your cell will die.

-pcrman-

QUOTE (scolix @ Jul 11 2007, 04:36 AM)

Too much lipofectamine can kill cells upon transfection. You need to optimize it. May be you could increase cell confluency or reduce lipofectamine while transfecting.

Yeah, I know, next time I'll try to use Oligofectamine, a guy in my lab use it and it works very well.

QUOTE (pcrman @ Jul 11 2007, 05:49 AM)

RPE-1 cells are Human retinal pigment epithelial cells. They are more "normal" than HeLa cells. They grow in DMEM-F12 supplemented with 10% FCS. The transfection mixture is prepared in Optimem-1 serum free. The growth medium is DMEM-F12 supplemented with 10% FCS but without Peni/Strepto (antibiotics).

Thanks for your answerings

BTW this site is cool !

-Kooothor-

Hi Koothor,

So the problem is the cytotoxicity of the transfection reagent. I found that Qiagen's HiPerfect reagent has the lowest toxicity to cells for siRNA transfection.

-pcrman-

We have used Qiagens SuperFect, and I have seen that it works very well. It is non-lipid based, so you can have serum in the medium, and with Superfect anti-biotics are not a big problem either.
But as I have seen, after a lot of trial and error, the suitability of any reagent depends a lot on the cell line under consideration.

-Gallus gallus-

Hi,

I've had the same problem with Effectene from Qiagen once. We have been using Effectene for a long time, mostly with good results. But after getting a new kit suddenly all our cells started to die within 16 hours after transfection. After talking to Qiagen we got a new kit, from a different batch and the problem was gone.

-boxfish-

QUOTE (pcrman @ Jul 11 2007, 06:40 AM)

I found that Qiagen's HiPerfect reagent has the lowest toxicity to cells for siRNA transfection.

I think that's depending a lot of the cell line.

QUOTE (boxfish @ Jul 11 2007, 08:07 AM)

I've had the same problem with Effectene from Qiagen once. We have been using Effectene for a long time, mostly with good results. But after getting a new kit suddenly all our cells started to die within 16 hours after transfection. After talking to Qiagen we got a new kit, from a different batch and the problem was gone.

Ok, I'll try to get a free new kit too

-Kooothor-

I use oligofectamine 2000 reagent for RNAi in RPE-1 and I have no problems of mortality. I use 4 microlitres of oligofectamine in a 10 cm well. Final volume 1 ml.

-bioxesus-