RNA Isolation using Trizol - Getting an additional band and v.low 260/230 (Dec/15/2005 )
I have been trying to isolate RNA from rat mammary glands using the TriZol method. Over the last 2 preps I find that my 260/230 ratio is <1.2 and I get a distinct band at about 1 kb ( I will try to attach the gel pictures ASAP). I quantitate my RNA using nanodrop and in the graph part I notice a distinct shift in the curve towards 270nM. The curve appears flatter than usual.The nanodrop manual says that this may be due to co-purified contaminants.
I am not sure how to correct this. I have never had this problem before when I worked with the same set of tissues. I asked the microarray facilities manager and she says the 270 shift is due to TRizol contamination
I need to use the RNA for Real time. SO here is the question
1. ANy random guesses what this contaminant is...other than the obvious..DNA
2. If so, how do I correct this before I use the RNA for anything else, if it is only DNA contamination, I can use DNAse , but I am not sure how to correct the TRizol contamination?
3. ALso, will it take care of both DNA and Trizol contamination if I used RNeasy column
Valuable opinions and advices please.
Thanks in advance
low 260/230 ratio indicates contamination with one of the following: organics; carbon rings (eg phenol); alcohol; high salt
This will have nothing to do with DNA
Trizol contains all of the above. Cleaning up with an RNeasy column will definitely help. Make sure you do the extra 2 minute spin at the end(before elution), or you might get ethanol/guanidine carrover, putting you right back where you started
[I did as you suggested and it worked perfectly...