I am extracting RNA from Zebrafish tissue (Brain, Testes, Embryos etc) using standard Trizol total RNA extraction protocol. The RNA concentration and yeild are good. Checked on Qubit. RNA quality is very good too (eRIN >8). Checked on Agilent TapeStation.
On checking the RNA using a Nanodrop, it was found to have peaks indicating Trizol/Phenol contamination. How will this affect downstream steps for RNA-Seq library prep? The sequencing platform does not even do a Nanodrop check.
If anyone has similar experience or potential solutions, please let me know. Thanks.
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
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA