RNA quality from total blood - (Dec/12/2006 )
I'd extracted total RNA from blood sample, and when I've check it quality, I notice that my RNA wasn't good - OD 260/230 between 0.5-1.0.
I am using TRIzol reagent for RNA isolation, so I would like to know what should I do to increase my RNA quality? Does a chloroform wash following RNA isolation (aqueous phase from organic phase) really work well?
Please, does anyone could help me?
With Trizoll reagent i always find a bad ratio of OD 260/230, too! For example: 2 total microgram of RNa gave me OD260/280 of 1,8 and OD260/230 of 0,6.
But I think it depends also on the total RNA quantity; however Invitrogen says that from Blood, Trizoll reagent is not the good choice! It is usefull extract RNA with column system!
well i use trizol for a while and my 260/230 are always playing with 2
are you sure that :
- you don't pipett phenolic phases
- you wash efficiently with ethanol 70 to get rid of salts
- you dry your RNA pellet enough
add 1ml BuOH.
Homogenize well by handshaking (avoid vortex) till you see a clear solution
pellet 1' 2000rpm
you should see a pellet or a least kind of a bubble which represents the aqueous phase.
So pipett the BuOH (upper) and redo the procedure.
If you see a clear pellet, spin without pipetting the buOH by 12000g at least for 5' (attach better the pellet)
then do 2 washes by EtOH 70%
the butanol is very hydrophobic so it can efficiently pellet all nucleic acids
on the other hand, it needs to enhance washes by 70%EtOH
Thanks, apemaia and fred_33! I'll try BuOH precipitation and check it out. Thanks!
Another question: I have some RNA samples with these bad OD 260/230 rates; is there something I can do to recover them?
Just clean the samples up using a column
Thanks again you all!