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No difference between transfected and untransfected cells - Using eGFP siRNA on GFPHEK293 cells usign flurimeter (Apr/21/2010 )

Hi all,

I am facing a great difficulty in my transfection experiment. I am using HEK293 cells stably transfected with GFP (Gentarget). I am plating cells at 10000 cells/well in 96 well plate and transfecting with 1-10ug/mL LF2000 with 10 and 100 nM siRNA (Dharmacon). I am doing the end point assay usign microplate flurimeter. Initially I saw high background even with optimem which fixed using water (nuclease free) as the media during reading. So at the time of assay, I aspirate media and replace with water just before the reading (488 exc/ 520 emi). (During this time some cells come out of the surface). I checked for flurescence after 24, 48 and 72 h but see no knockdown. I checked the siRNA sequence and it matches with the gene inside the cells. Its absorbance peak is good so it is not degraded. The cells do look flurescent under flurescent microscope when untreated and there is not much difference (visually) when treated with lipoplexes.

I do see some dead cells in untransfected well (trypan blue staining). I am incubating cell at 37C, 5% CO2, clean incubator, media DMEM+10% FBS + 5% L glu + pen-strep) Should this be the reason? Should I change the assay method? Should I use higher si concentration? Cells are also scraped off the surface when I changed the media to water before the readout.

Is it that GFP is too stable. Has anyone worked on these cells?

Please help.

Thanks in advance
Mamcy10

-Mamcy10-

Thoughts initially, of a technical nature - Use PBS rather than water as your fluorescent reading medium. Try using Lipofectamine RNAiMax rather than LF2000, it does give better siRNA transfection. Good peaks on a spec don't mean that it is not degraded - try running some dNTPs through the spec some time.

Do you know what promoter is used for the expression of GFP in the cell? I presume it is one of the viral ones such as SV40 - in which case, it will be massively overexpressing, so your siRNA might be working, just there isn't enough of it. Do you (or someone nearby) have a siRNA against another gene that you can use to check that the transfection is working?

Why are you trying to knock down GFP expression? If it is to remove the GFP from the cells so that they behave normally, then just get some normal HEK293 - it will be much simpler.

-bob1-

Hi Bob,

Thanks for your reply. I am using LF2000 as a positive control and want to compare the efficacy of my new formulation to LF2000. My purpose is to prepare effective formulation for siRNA delivery for which I am doing the gene knockdown assay.

I think promoter is SV40. I did try scrambled control but there was no diff between GFP siRNA and scrambled control.

I recently tried facs and saw lot of dead cells in all the samples including untransfected cells. The stock from where the plating was done shows good cell viability. I dont know what hapened to them in 96 well plate. I had plated 30K - 50K cells/well. I tried even in 24 well plate with 2 X 10^5 cells/well but still lot of dead cells. Media here is DMEM+ 10% FBS, no Ab. For FACS I changed to CO2 free media with serum.

Any suggestions?

Thanks
Mamcy 10




bob1 on Apr 21 2010, 08:40 PM said:

Thoughts initially, of a technical nature - Use PBS rather than water as your fluorescent reading medium. Try using Lipofectamine RNAiMax rather than LF2000, it does give better siRNA transfection. Good peaks on a spec don't mean that it is not degraded - try running some dNTPs through the spec some time.

Do you know what promoter is used for the expression of GFP in the cell? I presume it is one of the viral ones such as SV40 - in which case, it will be massively overexpressing, so your siRNA might be working, just there isn't enough of it. Do you (or someone nearby) have a siRNA against another gene that you can use to check that the transfection is working?

Why are you trying to knock down GFP expression? If it is to remove the GFP from the cells so that they behave normally, then just get some normal HEK293 - it will be much simpler.

-Mamcy10-

Mamcy,

You might try transfecting the cells three times in succession- once every other day. This may help to knock-down the GFP and overcome the protein stability - if it is part of the problem.

Also, I recommend Genesilencer from Genlantis for siRNA transfection. It works great for HEK293 cells.

HEK293 cells are generally easy to transfect, although stable transfection might change this characteristic.

It is very typical for HEK293 to come off the substrate; they usually do not stick very well. We are very careful when handling them: we try not to bump the plates, and add/remove media very gently.

You might try NOT using the pen-strep, as this can interfere sometimes with transfection.

I hope these suggestions help.

Mike

-San Diego Mike-

Hi Mike,

Thanks. I would try these. Lets see how it goes.

Mamta

San Diego Mike on Apr 22 2010, 06:16 PM said:

Mamcy,

You might try transfecting the cells three times in succession- once every other day. This may help to knock-down the GFP and overcome the protein stability - if it is part of the problem.

Also, I recommend Genesilencer from Genlantis for siRNA transfection. It works great for HEK293 cells.

HEK293 cells are generally easy to transfect, although stable transfection might change this characteristic.

It is very typical for HEK293 to come off the substrate; they usually do not stick very well. We are very careful when handling them: we try not to bump the plates, and add/remove media very gently.

You might try NOT using the pen-strep, as this can interfere sometimes with transfection.

I hope these suggestions help.

Mike

-Mamcy10-

Mike,

Question for you. If I do successive transfections, should I change the media before each transfection? Alternately, if I do transfection at once and observe knockdown after 24, 48 and 72h, should I add more media after every 24h, to keep cells alive?

Also do you have any idea on the initial cell density (for successive transfections) and in case if I observe knockdown after 72h for 96 well plate

I would welcome if others have suggestions for this.

Thanks in advance.

Mamcy





Mamcy10 on May 2 2010, 08:29 PM said:

Hi Mike,

Thanks. I would try these. Lets see how it goes.

Mamcy

San Diego Mike on Apr 22 2010, 06:16 PM said:

Mamcy,

You might try transfecting the cells three times in succession- once every other day. This may help to knock-down the GFP and overcome the protein stability - if it is part of the problem.

Also, I recommend Genesilencer from Genlantis for siRNA transfection. It works great for HEK293 cells.

HEK293 cells are generally easy to transfect, although stable transfection might change this characteristic.

It is very typical for HEK293 to come off the substrate; they usually do not stick very well. We are very careful when handling them: we try not to bump the plates, and add/remove media very gently.

You might try NOT using the pen-strep, as this can interfere sometimes with transfection.

I hope these suggestions help.

Mike

-Mamcy10-