RNA extractions of olive leaves using trizol method (followed by purification with Phenol: cloroform: isoamylic alcohol 25: 24: 1 and precipitation with AcNa 3M) sometimes yield brownish-greenish coloured solutions. Measurements with nanodrop gives high 230 nm readings, which I found in nanodrop users manual to be "generic contamination". Quanitification with qubit fluorimeter yielded inconsistent results. Are they polyphenols? How can I get rid of them?
I usually purify my RNA from Trizol using a QIAgen RNeasy column. I follow the protocol for QIAzol clean-up. I have done RNA extractions from tissues with high levels of porphyrin that were carried over with the DNA. Perhaps you are getting carry over of chlorofyll, which is a porphyrin.
That's interesting. Do you know the absorption spectrum of porphyrin in the 200-300 nm range? After a first glance, I just can find the spectra in the visible range (400-700nm)
Did you tested your samples for downstream applications (such as cDNA synthesis + qRT-PCR)?
Because you could have good results also with contaminations of greenish-brownish pigments. For example we extract RNAs from mosquitoes tissues and the head give every time a purple-red pigmentation of the RNA pellet (even in the resuspended pellet the solution is pinky). In the heads extractions I noticed every time abnormally high readings at 230nm but the qRT-PCRs or normal PCRs are fine exactly like other unpigmented tissues we use daily.
If the brown color comes from polyphenols, it might help to add Polyclar AT (that's a special kind of polyvinylpolypyrrolidone from Serva) during the grinding. It's insoluble, so it go into the pellet with the cell debris and will not be a problem later. We normally add x mg of Polyclar AT to x mg of plant material.
I second the use of some sort of polyvinylpyrrolidone in the extraction buffer. These are high-molecular-weight molecules that complex with polyphenols. There are numerous papers describing RNA isolation from phenol-rich tissues, and PVP is a recurring theme. I don't know how much (if any) PVP is in TRIzol, but don't be afraid of ditching the TRIzol and doing the extraction the old-fashioned way if you need higher quality.
Thank you all. Using PVP seems a good choice, although I have my samples already grounded so I must find how to merge PVP in the trizol extraction.
Dear Galip, phase separation seems to be normal. Thank you for your advice, maybe I try ethanol wash before doing a new extraction protocol.
Dear Paolo, I didn't try qPCR since I need accurate concentration determination in order to perform the dilutions prior to qPCR. No good concentration measurement, no dilutions, no qPCR.
Thank you very much Mr. Spencer for your valuable contribution and for the attached papers.
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