2 years ago
Paul G. Macon4
Hello! I used Trizol to extract total RNA from in-vitro cultured bacteria (1 X 10^8 cells). After phase separation, I mixed ~0.4 ml of the upper phase which contains RNA with 0.5 mL cold isopropanol. However, the amount of RNA when measured in Nanodrop was very low. In addition, the ratio between 260 and 230 was around 0.1 to 0.5. Is there a chance that my sample was contaminated by the Trizol reagent? When I collected the aqueous phase I made sure to not touch the lower phase. What should I do?
In my opinion the problem was using cold isopropanol. When used cold it can precipitate salt as well as RNA very effectively. Trizol reagent is also containing guanidium salts which is the reason for your poor 260/230 ratio. Collect the aqueous phase by following the protocol and add an equal volume of room temperature isopropanol. If after obtaining clean RNA, by following the rest of the protocol, you still have low yields you can try to increase the cell count.