The aqueous phase goes to the bottom when its density is higher than that of phenol-chloroform phase. This happens when you use Trizol on a sample which contains some salt at high concentration or a large amount of water-soluble organics. I saw such a phase inversion when extracting RNA dissolved in very concentrated Tris buffer.

By the way, if you do not want to pipet the aqueous phase from underneath the phenol-chloroform phase, you may try diluting your RNA sample with water prior Trizol extraction. Alternatively, you may desalt it by gel-filtration or using centrifugal ultrafiltration membrane units or by dialysis, and then proceed with the extraction.

thank you sir.

in my project, extraction doing from tissues.

Also, several times during extraction, when the aqueousphase went down, we used the upper phase. And by the way, we got a high concentration from it with nanodrop. Can this be normal???

It might be phenol. Phenol absorbs UV pretty well at 260 nm even so the maximum absorbance is at ~ 270 nm. You may take a look at the entire UV spectrum of your sample between 220 to 400 nm, it may give you an idea if you have RNA or something else in it.

hello sir, I can do it. RNA concentration in nonadrop was be 2107 ng/ul

Thank you so much