TRIzol wasn't homogenized with sample? - (Oct/14/2008 )
I'm extracting RNA from erythrocytic parasites using TRIzol. My sample is the frozen, packed RBC.
Yesterday I ran into a problem. My sample seemed not to be homogenized with TRIzol. Usually I'd pipette and vortex until sample and TRIzol are well-homogenized, but this time it looked more like colloid than well-homogenized solution. When let stand for a while, those unsolubled sank down to the bottom of the tube.
I thought it could be that I didn't break the cells thoroughly, so I freeze-thawed the sample-TRIzol mixture. That didn't work.
I'm puzzled already. I've been working with this kind of sample, but this is the first time I saw something like this.
I'm quite concerned with RNA yield, so I'm not sure if I should ignore it and proceed.
Could it be anything wrong with my TRIzol? Any suggestions?
Thanks in advance!
I think you should lyse your RBC before do Trizol extraction using NH4Cl or any red cell lysis buffer.
Thanks for suggestion.
I thought TRIzol itself should be just enough to break the RBC, and it was for what I've done so far.
I decided to go on afterall, and ended up with nothing. I guess I'll try again.
Trizol should be able to break down anything...
Maybe your cells were pelleted with too much force, whitch caused the pellet to be really compact, and thus insoluble.
I'm not sure that lysing cells before extracting RNA is a good idea. You could (in my opinion^^) either loose RNA, let RNAses of the RBC free (and thus loose your RNA) or contaminate the Trizol reaction with any product that are in you lysis buffer.
That's exactly why I'm hesitated to break cell before adding TRIzol.
Anyway, I tried increasing TRIzol volume and it seemed to work. Maybe the cells are indeed too compact for recommended TRIzol volume.
However, RNA yield is pretty low. I don't know if it's caused by the altered ratio of TRIzol-sample. :p