Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!

orna's Content

There have been 3 items by orna (Search limited from 10-August 19)


Sort by                Order  

#8489 problems in getting stable clones

Posted by orna on 06 November 2004 - 09:47 PM in siRNA, microRNA and RNAi

Hi,
I had transfected my RAW cells with pRetroSuper-hygro that contain siRNA cassette.
After two weeks I got cells that survive the selection with hygromycin.
When I did a western blot I found that the cells expresses the protein that was supposed to be knockdown.

I know for sure that I got an efficient siRNA for my gene. It works in other cell lines. It works in reporter gene assays.
I didn't check the RNA levels and the western signals are specific.

What do you think is the reason that the cells resistant to the antibiotic but do not express the siRNA?

Is their any chance that the vector partially integrated to the genome?
Or may be the gene I'm trying to silence is too important to survive that the cells manage to express the hygro resistance gene and not the siRNA?

The reason I'm asking in this forum is because I don't think that I have problems with my siRNA.
Did you experience something like that?

Thanks a lot
Orna



#8302 stable clones - but expressing the gene! help!

Posted by orna on 31 October 2004 - 09:51 PM in siRNA, microRNA and RNAi

It works in other cell lines. It works in reporter gene assays.
I didn't check the RNA levels and the western signals are specific.

Is their any chance that the vector partially integrated to the genome?
Or may be the gene I'm trying to silence is too important to survive that the cells manage to express the hygro resistance gene and not the siRNA?

thanks
Orna



#8296 stable clones - but expressing the gene! help!

Posted by orna on 31 October 2004 - 05:55 AM in siRNA, microRNA and RNAi

Hi,
I had transfected my RAW cells with pRetroSuper-hygro that contain siRNA cassette.
After two weeks I got cells that survive the selection with hygromycin.
When I did a western blot I found that the cells expresses the protein that was supposed to be knockdown.

I know for sure that I got an efficient siRNA for my gene.
What do you think is the reason that the cells resistant to the antibiotic but do not express the siRNA?
Thanks a lot




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.