How to get rid of guanidinium contamination - RNA purfication (Aug/24/2005 )

Hi,
I am extracting RNA from HUVEC cultures and am having problems with guanidinium contamination. I have used Trizol and also the Qiagen kit and am still having problems. I am using the glycogen carrier because my cell counts are low but 260/230 ratios are still well below 2.0 (260/280 is fine).
Any tips on how to get rid of it and if I cant will it interfere with cDNA syn thesis and TaqMan PCR.
Thanks

I don't really know how to get rid of the contamination. However, I have never had an issue making cDNA from it and using it in real time PCR. Try it out!

Sheila

How do you know that you have a guanidinium problems?

Daniel

DNA sequencing software

Hi,
I want to ask the same that Daniel: How do you know that your sample is contaminated with guanidinium isothiocianate?

guanidinium isothiocianate is a denaturant salt, so it could damage reverse transcriptase in your cDNA syntesis.

I am sure that you have thougth about it, but let me suggest that when taking the aquous upper phase stay as far as possible from the interphase.

Since the contaminant compound is a soluble salt you could try to wash your RNA many times in EtOH 75%

I would hazard a guess and say that you probably have phenol contamination, rather than guanidinium contamination. If you are willing to endanger a sample (should be fine), have a smell of it, if it smells sweet, it probably still has a tiny bit of phenol left. NOTE Don't smell a bottle of phenol to find out what it smells like, it is quite toxic in the pure states, but solutions less than 0.5% are fine apparently as many lip balms have it at that conc.

Rextract, starting from the chloroform stage is my suggestion.

Good luck
Bob

guanidine carryover is quite common with QIAgen (and other silica) kits. QIAgen themselves will tell you so