RNA isolation by Trizol - (Nov/27/2008 )

Just a few questions about RNA isolation. 1.) I used 1-2ug of total RNA on a 1% agarose gel and stained with EtBr. How is the quality of the RNA? Do the bands above 28S refer to genomic DNA? 2.) The samples were collected by my colleague. She collected the samples and then stored at -20C for a week. After thawing the samples, the trizol solution was not clear, with milky-like cloudy clumps. Have you come across this? Why does this happen?? She asked me to use vortex to mix it. I am wondering if vortex will cause the RNA with genomic contamination.

Thanks a lot!!

Samantha

for me, the RNA looks ok. the bands above the 28S bands should be mRNA (or some bigger fractions of rRNA). genomic DNA is the white stuff in the wells of your gel.

two possible causes of gDNA contamination: too much sample material used or too much of the aequous phase removed.

mixing the trizol should be no problem (and is even required in the protocol, i think).

Hi, neigbour lab use trizol for RNA izolation. They do not freez it, sample can by storet in trizol for week in 4 C and izolated withot decrease in quality, they told me. They had the same experiences with freezing milky -liky but I dont know how they solved it
good luc

Hi Sam!!
Based on my experience on RNA i can tell you that milky-like clumps are quite normal and i think these are because of rest of the inicial sample without total homogeneization, but they dont interfere with rna isolation. you don't say if the extraction was from tissue or cells but in the second case I would recommend you to use liquid nitrogen to disrupt the tissue before and after adding trizol and pulverization in a frozen mortar. I don't think vortexing the sample is going to cause bigger dna extraction; neverthless you must use a dnase treatment kit to treat your rna samples after extraction and be sure of that.
One more thing is that i would strongly recommend you to put the samples at -80 when storing them because i have measured samples stored for some days at -20 and they have less concentration than at the beggining, and they tend to degradate with multiple thawings.
I hope to solve your doubts.
Rach el

Thank you very much! So is the quality of RNA fine?? How come some bigger rRNA and mRNA appear on the gel? Because for most of the time, I don't see these extra bands (the bands above 28s)

Also, they are cell line samples.

Samantha

I think you should store your samples at 80C. But if degradation of RNA you wwould see below your 18S band. It's probably sheared genomic DNA or total RNA. Do you follow the protocol extraction carefully, ie all centrifugation at 4C? If the precipitation steps are not cold enough , the RNA/DNA may not separate properly.