FITC-conjugated siRNA - (Apr/01/2005 )
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!
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 "HiPerFect" (Qiagen).
I hope I could give you a few hints
Thank you, Bomber. I indeed get some hints from your reply now.
I am going to transient transfect the FITC-conjugated siRNA into NMuNG cells. Do epithelial cells work great with siRNA transfected by using vectors?
Unfortunatley I do not have any experience with your cells.
If there is no one who can help you about about the vector transfection and in case you received the vector from a company I would call the technical assistance service of the company, sometimes they provide nice protocols or are even experienced with the cells you are working with.
Let them provide some information to you .
For a transient transfection siRNA is nice as you don´t need to clone anything. If you need to get some stable clones I can´t think of any other method than using a vector-system.
Anyway it´s always good to check that your siRNA is working properly and you are targeting the right region in your target-mRNA so a first experience should be a transfection of siRNA (maybe even when your siRNA is published for other cell types): better see the effect in your cells .
Cheers and good luck
Thanks for your help, I do get idea what I should do.
Cheers and thanks again