Phenol contamination isn't good for downstream applications, generally speaking.

The quickest and least-fiddly method for removing phenol contamination (in my experience) is a basic chloroform extraction. Warning: you will lose some RNA (ca. 30%, typically).

Make your RNA sample up to a manageable volume with DEPC H2O. I generally find 500ul to be sufficient.

Mix this 1:1 with chloroform, and vortex thoroughly: you ideally want to vortex till it turns cloudy, ensuring comprehensive mixing of any water-dissolved phenol with the chloroform (the two phases will start to separate out again pretty quickly, mind).

Centrifuge (chilled benchtop, 4deg, 13krpm, 10min), take the aqueous phase (upper).

Precipitate RNA as usual (room temperature isopropanol for 20mins as per TRIzol protocol is fine).

Wash with ice cold 70% ethanol 2x

Dissolve in a suitable volume of H2O.

Hi Roy,

I had similar problem when I used the standard Trizol protocol for total RNA extraction from fish tissues (gill, liver, kidney from rainbow trout). In downstream gene expression analysis by qPCR sometimes we found significant differences between replicates of the same tissue, (also when collected from the same animal!). We found that Phenol and/or guanidine salts in the extracts affected following RT and PCR steps.

Then we shift to RNA extraction protocols, always Trizol based, but with final purification in silica column (Qiagen miRNeasy/ RNeasy universal mini kit) and inhibitions and SD between replicates was lowered.

Gook luck,

best regards

Alessandro

Thank you for your replies.

@John

I agree with you. I think I will have to reprecipitate and do an extra wash. I usually do not vortex after the initial tissue disruption stage. I think it breaks fragments and lowers RIN scores. Also critical if pulling out mRNAs using polyA beads. For single embryo samples, the yeild is already too low (~100ng), therefore I am not sure if I can afford to lose more by further washing.

@Alessandro

I had tested the Qiagen miRNeasy kit initially and I found that the yeild and quality were similar to Trizol method. And since the Trizol approach was significantly cheaper, I went with that. But back then, I didn't have the Phenol contamination issue. Or perhaps I didn't check it then.

DO NOT wash RNA pellets if you are analyzing small RNA.  This will selectively loose some small RNA and this issue caused a paper in Narry Kim's lab to be retracted (see link).

What I do is resuspend the TRIzol pellet into a large volume of water (400microliters), transfer to a new tube, and then proceed with an ethanol precipitation without a wash. Add glyco-blue or another co-precipitate if you have low RNA levels. I've done all my RNA extractions (specifically for miRNA qPCR) with TRIzol, and doing this second ethanol precipitation gives me great RNA quality by 260/280 and 260/230nm absorbance ratios.

http://www.narrykim.org/static/articles/2012/10.1016-j.molcel.2012.05.036.pdf

An alternative to chloroform extraction and precipitation would be a Qiagen RNeasy MinElute Cleanup Kit. You can use this to clean and concentrate your RNA, removing phenol and other contaminants such as salts and inhibitors.

To avoid getting the phenol contamination in the first place, make sure you spin long enough when you do the phase separation step during the trizol extraction, handle the tubes carefully and remove the clear top phase containing the RNA slowly. Do not try to be 'greedy' and take the whole lot - you will get genomic DNA contamination from the interphase layer and also some of the phenol. Always leave some of the clear top phase to avoid this happening.

You could also use columns such as Direct-Zol from Zymo instead of precipitation to clean up and elute the RNA after trizol - this may help to avoid the phenol carry-over.