Problems with Trizol - (Nov/19/2005 )
I have been doing some RT-PCR experiments from mammalian cells (tissue culture, adherent) and I have some problem that has been driving me crazy and figure this well-informed forum can help me out...
I am using TRIZOL from Invitrogen to isolate my RNA as this is an easy and reliable method of RNA extraction. However after I mix Trizol with chloroform, take the upper phase and isopropanol to it, something strange happens. After I spin my samples, instead of getting an RNA pellet I get a "bubble" at the bottom of my tube. What I mean by bubble is that it looks like another lower orgnanic phase that is separate from the aqeous/isopropanol mix. The size of this droplet is about 30-50ul.
Funny thing is if I remove the supossedly aqeous phase and spin down - no RNA pellet. If recombine and mix, then spin down - NOW iget my RNA pellet. I go through the normal steps of washing the pellet, redissolving the RNA, DNase treatment, and RT. When I PCR for GAPDH or B-Actin I get horrible results that are widely variable. This procedure has been done many times in the lab and even GAPDH bands are the norm...
Any help/suggestions are appreciated,
One of your components must be incorrect.
In order to identify it, do control experiments:
1) new batch of Trizol
2) new batch of Chloroform (are there additives in that chloroform, btw?)
3) No sample control (Trizol and chloroform only)
Perhaps you underestimated the volume of sample? Respect the ratio sample vs Trizol.
I've had the *exact*same thing happen to all of my samples since getting a new bottle of trizol. I did an experiment in which I tried using an old bottle of trizol and our new one, and found that only the samples I used the new bottle with had an extra organic phase form in isopropanol precipitation. I ordered another bottle of trizol (from a different lot) and had the exact same thing happen. What was your lot number? I'd be curious to know if it's the same as one of mine. Sigma makes a similar product, so I'm going to try that. Let me know what you find.
I found this old thread trying to get some help on what appears to be the same problem. Every time I use Trizol I get a bubble after isopropanol precipitation, and no sign of a pellet. Often the pellet appears if I aspirate the isopropanol but leave the bubble, and clean with 70% ethanol - however, my pellets are poor, not well packed, and often dissappear when I lose the ethanol.
this seems to be limited to a single bottle of Trizol: has anyone followed this up, or contacted invitrogen about it? I'm going to contact technical support, as I've lost 50-80% of my RNA from a lot of experiments so far.
PS - the bubble appears when the extraction is performed in the absence of any cell extract - seems definitely to be the trizol.
i think that your first centrifugation is not enough long. That drives tiny rests of phenolic phase to get pellet only in second spin.
If you add a hydrophobic compund (enthanol), you're able to dilute the phenolic bubble in it, and that allows the RNA to be pelleted.
You may do a chloroform-only step after pippetting the aqueous phase. Phenol will get in it, and as chloroform separates better than phenol from water, you'll get rid of phenol.
thanks for the advice guys. I'm still not sure about its identity, it isn't choloroform because choloroform alone doesn't form a separate layer in isopropanol when we tested it. We do the chloroform extraction twice and spin for 10mins, so I was sure it wasn't phenol, but maybe it is - my most recent PCRs have not worked, and phenol inhibits PCR!
Still, we have been careful, and this is a recent problem which just makes me wonder about possible contaminants in the Trizol. In my most recent experiments, the bubble appeared as before at isopropanol precipitation (and the size / appearance is very consistent), so I took isopropanol off with a pipette instead of decanting. Then did the ethanol wash - still seemed to be the extra layer, though I did get some pellets - not all, but some. A260/A280 looked great, but the PCR didn't work. I'm assuming that is because there is some of this residue left because the isopropanol wasn't decanted.
Frustrating! Hopefully sorted by a fresh bottle of Trizol on Monday!
keep us informed for the results i wish you'll fix the problem
for those who may be interested, an update. I have replaced my Trizol with a fresh bottle, and the "bubble" problem has dissappeared. My pellets are now clear and visible after isopropanol precipitation, so I can no longer blame my tools if I lose a pellet!
I just thought I'd let you know, and maybe save some others the same trouble. For reference, my lot numbers were:
problem : 1298919 (200uL)
new batch: 1317023 (100uL)
Be warned though, I havnt run any PCR with my extracts yet, so its not 100%, but whatever the contaminant was has gone. I've spoken to Invitrogen about trying to get something back for all the PCR reagents etc wasted!
Guys, in this kind of situation get straight on to invitrogen and ask for a replacement lot free of charge. They will be more than happy to help