Problems with Trizol - (Nov/19/2005 )
Does anyone know why or how this can happen?
I think I am experiencing the same problem. I have changed all the other reagents, am not doing anything else differently in my extraction.
I am ordering a new bottle of trizol.
Still, I would like to know the theory behind this, if anyone has one?
I am new here and I registered because I read this topic and I met the same problem last week. I isolate total RNA with Trizol (from Qiagen) and then perfom a chloroform separation, isopropanol precipitation and then an ethanol wash.
Last week I added a poly-A carrier and isopropanol, then I spun down and I get a bubble at the bottomn of the tube instead of the normal gel-like pellet.
I remove carefully the isopropanol supernatant, then add the ethanol and spun down extensively. And then I could see the pellet!!
So I do not know yet if RT-PCR will work... I will see and let you know.
But all I know is that with the same bottle of reagent I didn't get this bubble before... so could it really come from chloroform? or Trizol? bad manipulation?
thanks for the coming explainations
We are still trying to work out the cause of our 'bubble' problem. We also replaced the TRizol and replaced the Isopropanol and still had the bubble issue.
Fortunately we have found now that it seems quality RNA can still precipitate despite the 'bubble".
If we work it out, I'll let you know.
We think we have discovered the cause of our 'bubble' problem.
It seems to have been using too much RNA later in the initial tissue disruption and carrying it over to the homogenisation.
Even though we kept the ratio of trizol to sample at 10:1, it seems that if there is too much RNA later carryover, it can cause a 'bubble' at the precipitation stage.
It seems to me that we are looking at separate problems: I performed side-by-side experiments with 2 lots of Trizol - 1 had the "bubble", the other did not. Further, I had the bubble when I ran the procedure in the absence of any RNA - I just used the solvents. Therefore I am 99.9% sure it was a contaminant in the Trizol, and Invitrogen replaced the bottle with no problems - fingers crossed.
However, if you have solved the problem that is the most important thing! For anyone else with the same problem, I would replace the solvents / Trizol as the first step. My chemistry isn't good enough to guess at what the contaminant could be!
there is sections thay may apply in your case
- RNA may have been speed vac or lyophilized after the last ethanol precipitation. RNA that has been dried completely has decreased solubility. Additionally, if excess centrifugation speeds (speeds greater than 12000 x g) were used, it is harder to solubilize RNA/DNA.
- RNA pellet may not be completely solubilized. To increase rate of solubilization, pipet repeatedly in SDS or DEPC-treated water, then heat to 50-60 C. Sample may also have been rich in polysaccharides or proteoglycans. If so, isopropanol precipitation step should be done with 0.25 vol isopropanol and 0.25 volume high salt solution (see section on Samples with proteoglycans and/or polysaccharides).
- Cells were washed prior to the addition of TRIzol. Washing cells before the addition of TRIzol increases the possibility of mRNA degradation.