I am trying to perform trizol RNA extraction on mouse skin samples. After addition of chloroform for phase separation, the top aqueous layer remained very pink - I think from Trizol contamination. However, we progressed to adding isopropanol to precipitate the RNA. No pellet was seen, even though the same batch on a previous day showed good concentration and purity of RNA extraction.
Is it possible to repeat the chloroform step again at this point after isopropanol addition to try to further remove trizol contamination? The samples still look very pink.
Any other suggestions would be greatly appreciated.
Thank you very much.
you should use 70% ethanol to purify and precipitate the RNA
I agree with Dr. Mahmoudpour, but you can change the protocol for any kit. Trizol in certain cases will not give you a good purified RNA, it may be that a drag of impurities occurs.
When I did this protocol I washed twice with cloroform before to go for next step.
The fact that you didn't see pellet with isopropanol doesn't mean you don't have it, sometimes it remains "invisible" until ethanol step.
In addition you can use a RNA carrier such as glycogen to improve your RNA precipitation, it also switches the pellet colour to white improving its visibility.
If your aqueous phase is remaining pink, I wonder if there's an issue with your separation? How old is the chloroform that you have? Could you see an opaque white intermediate layer? If not, then the separations aren't happening correctly and no amount of repeats will help. If you are, then you're right that the TRIzol (phenol) is contaminating your aqueous phase and you will need to work out why this is occurring if you want successful, clean RNA preps.
How much tissue are you adding TRIzol to? And how are you preparing the skin? Are you powdering it with liquid nitrogen? Are you adequately mixing the TRIzol and chloroform by tube inversion for 15 seconds? How long are you centrifuging the sample for following chloroform addition?
As has been stated by others, just because you don't see a pellet, it doesn't mean that there's no RNA there. But I think you need to understand what's happening to your aqueous layer.
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