I would like to perform a skin penetration assay in Franz diffusion cells, using liposomes containing Cy5-labeled siRNA. Besides looking at the penetration capacity of my liposomes, I'm also interested in the transfection capacity (by means of qPCR). now I was just wondering is it possible to do an RNAi experiment using frozen/thawed skin tissue? What about the RNA quality?
Hi Eline,
how do you store your skin tissue? normaly by -80°C there should be no problem with your RNA. You can test it with an RNA chip if you have than you will get also some information about your RNA quality. If not you can run your RNA on an 1% Agarose gel (clean everything before). Your RNA is ok when you see two big bands (http://cmg.iupui.edu/cmg/Agarose1.htm). Good luck
Dag Eline,
You'll find information to assess the integrity of your RNA in this publication. Note the authors from the University of Ghent. The publication is also important to perform RT-qPCR in general.
Storing tissues in the -80°C or in liquid nitrogen is the best way to rpeserve RNA if you cannot extract them immediately.
I don't understand your question about doing RNAi with forzen tissues? you mean do RNAi, freeze the tissues and anaylze them later, right?
Hope it helps,
Salukes
Gérard
I also did not understand your question. I think you are referring to in vitro skin penetration studies of RNA loaded in liposome. You can monitor its skin penetration using fluorescent label RNA (fluorescence microscopy) or extracting it and then analyzing it by PCR. If you want to verify its specific antisense activity and transfection, you can do it in cell culture or in vivo, because the frozen skin or even the recent dissected skin from animals or human do not maintain it metabolic condition during the permeation experiment. I hope this explanation can help you.
@Maria: Yes, this was the set-up I had in mind.
Thanks a lot for these answers.
- Badges
- Science topic