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Position: Discussion forums > Bioscience biotechniques discussion forums > Cell biology and histology methods forum
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FITC-conjugated siRNA
Posted by: doof (IP Hidden, New member, 1)
Date: May 27, 2005 01:11PM
I have two questions need for your help.
The first one is what the general transfection method is while I handle with FITC-conjugated siRNA? Make a construction using appropriate vectors, transfect via Virus infection or something else? The second question is what purpose of FITC is applied here? Thanks for your help!
Re: FITC-conjugated siRNA
Posted by: Susan Denner (IP Hidden, Unregistered user, )
Date: May 27, 2005 01:28PM
Hey,
I´m not an expert in siRNA etc, but anyway maybe this will help you a bit: In general the method of transfection (e.g. siRNA, vector or virus) depends on the cells you want to transfect. There are cells that work great with siRNA others are difficult to transfect so it might be improved by using a virus. A second point might be: what would you like to establish- stable clones? or is it a transient transfection? The purpose of FITC is to measure your transfection efficiency. In my case I used "alexa fluor 488nm" labeled siRNA as a negative control to be able to do a FACS-analysis. "Alexa fluor 488nm" worked very good for my purpose. I transfected cells with "Gencarrier" (Epoch Biolabs), I found it's cheaper, extremely efficient and without causing much cell death. I hope I could give you a few hints Cheers & Have a nice long weekend
Re: FITC-conjugated siRNA
Posted by: mygateway (IP Hidden, Junior member, 10)
Date: June 1, 2005 12:10PM
The FITC conjugation of siRNA can be archieved by linkage chemical conjugation of FITC at 3' of one strand of the siRNA. So there is no way to construct shRNA vector or virus to express the FITC-conjugated siRNA. You will have to perform chemical conjugation reaction and then transfect the siRNA into your cell line, using siRNA transfection reagents available commercially.
The FITC-conjugated siRNA can be used in FACS study or minitor the transfection efficiency, cellular translocation by cytochemistry study etc.
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