discuss about northern for miRNAs - northern, miRNA (Dec/20/2008 )

Hi there,
I'm working on miRNAs for 2 years.recently I encountered problems with northern.
1.I noticed that in the transfer step, my RNAs were often over-transfered, for I can see many on the bottom 3M WATERMAN. Then I reduce the time for transfering ,But this time ,I got no signal for U6 as loading control. Is there anyone can tell me how to adjust the setup for transfering?

2.many refference talked about the LNA-modified probe. I also sythesized sevral (at 5'-OR every 2 or 3 residules).But they just didn't work at all. I wonder whether It's the problem in sythesizing or purifing (I synthesized in a Chinese corporation and purified by PAGE)

and the last I want to know the menbrane you usually used for northen.I surpose the Ambion Bright Star possitive charge nylon membrane doesn't work very well.


Thank you very much!

Hi,

Regarding transfer I use a Genescreen Plus membrane - positively charged.
I perform a 1h semidry transfer with constant current (20 - 25 mA with voltage set at 3x membrane size) in 0,5xTBE.

You can try and do such control: load a labeled (ex. with P32) RNA on a gel. Then, after transfer place each layer of the “sandwich” in phosphor-imager cassette, 3MM Whatmans, membrane and check the signal. If the transfer is optimal, only the membrane should give a signal.

It works for me

Good luck...