RNA extraction by Trizol protocol - I didn't get a good quality of RNA (Oct/26/2005 )
I've done RNA extraction from human brain tumor by Trizol protocol and I didn't get a good quality of RNA. I used to removing normal and necrotic tissue from tumor fragments before the proceeding.
When I run agarose gel 1,2% I didn't get 28S and 18S bands. Moreover, I saw two inespecific bands in the end of each run and DNA band in the beginning.
What could these two inespecific bands be?
What could I be doing wrong?
What do you think I should do?
PS: I attached photo of my gel.
use twice volume as recommended for better recovery
work quick and on ice
use filter RNA-dedicatedtips, and RNA dedicated 100% etoh, IPrOH and 80% etOH solutions (80%etOH prepared with DEPC treated miliQ water)
clean your bench with 0,1N NaOH
clean pipettes too
pre heat trizol to 50° and carefully pippetting up and down till getting a clear solution enhances drastically the quality
maybe use RNA later?