Problems dissolving TriZol RNA pellet - (Nov/10/2008 )
I was running RNA extraction on mouse stomach fundus samples using Invitrogen's TriZol procedure. I had a lot of problems dissolving the final RNA pellet in water. I ended up spinning the samples down and throwing out the remaining pellet. I tried adding more water and also heating it (which we usually don't do, but the TriZol procedure says to do that), but it didn't work. Our initial samples were around 50 mg. We used linear acrylamide to precipitate out the pellet, which may be the problem. We also subsequently found out our samples had TriZol contamination, would this affect the solubility of the pellet? Also, we ensured the pellets didn't dry out before dissolving in water. Does anyone have an idea of why we couldn't get the pellets to dissolve?
Martin
RNA is not very soluble in water, try dissolving in TE or TLE.
Will TE or TLE interfere with QRTPCR?
It's the contaminating denatured proteins that make the pellet insoluble. Just spin down the insoluble (you would lose some RNA trapping in the pellet) after soaking it for a few hours on ice and transfer the supernatant to a new tube. BTW please give AquaRNA a try.
Will TE or TLE interfere with QRTPCR?
Final elution with TE pH 8 is a good option because It even prevents problems derived from ethanol or isopropanol contamination during RNA extraction process. Moreover, It stabilizes solution pH, withn can alter 280nm absorbance --> you "Purity ratios" (A260/A280) will become more reproducible and accurate.
But you should keep in cosideration that if you want to perform RT-PCR with your RNA, TE buffer can affect this process. TE contains EDTA, a powerful chelant that captures divalent cations like Mg++. Magnesium is needed by DNA polymerases, so the RT-PCR reaction may be difficulted if the amount of RNA template is too big. I advice you to work with the lowest template concentration if you finaly decide to elute in TE.
I hope my answer help you.
Are there good ways to reduce the amount of contaminating denatured proteins, apart from being more careful when separating the aqueous phase after initial centrifugation with TriZol of the homogenized sample? Is this a common problem?