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Position: Discussion forums > Bioscience biotechniques discussion forums > Cell biology and histology methods forum
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trouble with siRNA transfection
Posted by: 4321 (IP Hidden, New member, 1)
Date: May 24, 2005 07:27PM
I have trouble with XtremeGENE siRNA Transfection Reagent from Roche.
I've tried different ratio and follow every step of the protocol but nothing happened (no knockdown of my gene). The sequence of my siRNA is correct and have been validated on other cell types. Your comments are much appreciated.
Re: trouble with siRNA transfection
Posted by: knewman (IP Hidden, Unregistered user, )
Date: May 24, 2005 07:50PM
I also have the problem with this reagent on my Vero cells. Later, I changed to RNAicarrier then I got a ~90% knockdown of my target gene using the same set of siRNA.
Re: trouble with siRNA transfection
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: May 26, 2005 02:27PM
Yes, try other transfection reagents as the kit itself is likely to be the problem, especially as you've troubleshooted it throughly. Some kits simply dont work well with certain cell lines. I've had success with siIMPORTER from upstate. It has very low cytotoxicity and can achieve close to 99% transfection. good luck!
Re: trouble with siRNA transfection
Posted by: PNAS (IP Hidden, Unregistered user, )
Date: May 27, 2005 01:27PM
Hello everyone,
I have difficulties establishing RNAi in Jurkat cells (non-adherent cells) with constructs that do work in several adherent cell-lines. I am using shRNA in the retroviral pSUPER vector. Does anyone experience the same problem and does anyone have an idea of how to solve it ? Thanks!
Re: trouble with siRNA transfection
Posted by: 5'-ATCG (IP Hidden, Unregistered user, )
Date: May 27, 2005 01:37PM
(1) Try chemically synthesized siRNA instead of shRNA expressing cassette especially in lympholytic lines - the inteferon effect will be lower.
(2) It's easier to transfect Jurkat with synthetic siRNA than a expression plasmid. (3) Transfection - for Jurkat cells, electroporation or Gencarrier-2 will be your 2 top-picks.
Re: trouble with siRNA transfection
Posted by: Jurkatmom (IP Hidden, Unregistered user, )
Date: May 27, 2005 01:43PM
I too am having similar problems in getting knockdown in Jurkat cells with shRNA constructs that are working in overexpression studies in 293 cells.
I have also tried transfecting siRNA versions but these also seem to be inefficient at knocking down the endogenous protein. I have just been using gencarrier reagent to transfect the cells I am getting good transfection efficiency (40% with shRNA and >90% with the siRNA) but no knockdown. I was just wondering if you had solved your problem and have any tips for me? Any suggestions would be much appreciated.
Re: trouble with siRNA transfection
Posted by: 3'-UTR (IP Hidden, Unregistered user, )
Date: May 27, 2005 01:52PM
Transfection of siRNA is a very tricky work and needs to be optimized for various cell types.
But there are other many things that might influence the outcome of a "knock-down" in different cells. These are for example: 1) different amounts of proteins that are needed for generation of a knock-down (for example RISC-complex) 2) the abundance of target mRNA seems to influence the efficacy of a knock-down as well. Although a certain "sequence" might work in many different human cell lines there is definitely no guarantee that it will work in another (I just made the experience!).
Re: trouble with siRNA transfection
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: May 27, 2005 02:11PM
If you use Gencarrier be especially careful and it has an extremely high toxicity to many cell lines.
Re: trouble with siRNA transfection
Posted by: Jurkatmom (IP Hidden, Unregistered user, )
Date: May 27, 2005 02:36PM
In my hands, gencarrier worked best among lipofectamine2000, fugene-6, GeneJuice, JetPET....in low toxicity and high efficiency. Lipo2000, fugene6 all showed much higher toxicity than gencarrier when loger exposure.
Re: trouble with siRNA transfection
Posted by: bighead (IP Hidden, Unregistered user, )
Date: May 27, 2005 06:43PM
Hi, Jurkatmom,
I am trying to transfect Jurkat cells with a GFP-expressing plasmid. I used lipofectamine 2000, X-treme, FuGENE with no good results. I obtained around 2% GFP (+) cells among the ~60% left living ones. My settings were: 0.4 million cells/well in 24 plate, with 1 microgram of DNA. When I add PMA the GFP expression reaches 5% after 24 hours. I also tried electroporation with the same bad results, only 8% with ~50% cell death. I used 5 million cells in 300 ul, 960uF, 240 V, gap 0.4 cm, normal medium. I've also heard good comments in other forums about GenCarrier transfection reagent, I would be much appreciate if you can share your experience with me. Thank you!
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