I am using the QIAGEN miRNeasy kit for total RNA isolation from tissue and serum samples as per manual instructions. However, I am facing issues with the phenol contamination and the 260/230 values are very low (0.8 - 1.2) in the cases of both tissues and serum. Also, after the phase separation step, there is a medium size layer of trizol on top of the aqueous phase which I suspect might be the reason for phenol contamination. What could be the possible reason behind this and how do I solve it? Please explain. The centrifugation is at 4C X 13200rpm for 15min -20min as per protocol.
Hi! I think you should confirm this by running a UV scan. Normally phenol contamination gives rise to something like a nub in the spectrum at 270 nm. Anyway, I suggest you can use diethyl ether to clean any trace of phenol. Just mix 1:1 v/v vortex, spin and put it at -80 for a couple of minutes. The diethyl ether doesn’t freeze so just take it out. Repeat once or more time. Hope it works!
Another option is to make a chloroform extraction of the "aqueous" phase (after the trizol extraction). The phenol goes to the organic phase (chloroform)so you will get clean extracts with better 260/280 ratio. For the extraction you can use one volume of chloroform: isoamyl alcohol (49:1). Usually this works well. Good luck!
Thank you for replies!!!
Not sure about using isoamyl alchohol as this is not recommended by miRneayt kit protocol. Also, about diethyl ether. I am extracting total RNA for microRNA profiling so I am not sure if above steps should be done or not....
After obtaining total RNA contaminated with phenols, I have performed total RNA cleanup protocol with QIAGEN kit which helps and pretty much improves the 260/230 values. However, this is also causing loss of upto 30% total RNA which I cant afford at this stage due to very small tissues. What my major confusion is that why aqueous phase is getting turbid with trizol as during my previous extraction steps with Trizol itself and QIAGEN kits, I have never seen any such issue.
Hi, I also used QIAGEN miRNeasy kit, for total RNA and miRs extraction.
I remember that sometimes, in particular with tissue samples, the problems lead in the amount of starting material, I need to used more QUIAZOL (for example 2ml) and than divide the samples in two tubes.
Another problems that I have is that if you do not shake very well the sample after the addition of chloroform, the phases are not well separated, I often shake and also vortex and centrifuge immediately after, if you see some probles after the centrifugation shake and centrifuges again.
Those are only my personal experiences, It is important to follow the other advices to clean your RNA samples but for the new ones I woul try to:
Pay attention to the amount of starting material and homogenation (at least for tissues samples)
Use pure chloroform (I used a bottle "RNA dedicated")
Shake very well or vortex for few second and the centrifuge immediately
I hope this help good luck!!!
Is not about isoamyl alcohol alone, but I suggest is an extraction (cleaning) step with a mixture of chloroform and isaomyl alcohol in a proportion 49 volumes of chloroform plus 1 volume of isoamyl alcohol (49:1). This is usually used for RNA extraction.
I agree with above comments. nature and amount of samples highly matters during RNA isolation. try to reduce amount of tissue and increase trizol amount.
Hope it helps!
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