Some help with RNA isolation using Trizol

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5 years ago

Paul G. Macon

2

Some help with RNA isolation using Trizol

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?

Experiment: RNA isolation / purification Bacteria - Gram positive Clostridium difficile

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**Luud Roorda** · Answer 1 · 2019-05-14T14:42:37+00:00

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.

**Gladys E. Ott** · Answer 2 · 2020-05-27T09:57:55+00:00

There should be clear bands on the agarose gel for the 16S and 23S rRNA if it's good quality. I also know we always used to use a TBE gel instead of TAE for RNA but I don't remember the reason/if it was actually important... Good luck!

**Dhr. L.S. Petitiaux** · Answer 3 · 2019-07-11T18:29:56+00:00

3

Great stuff, thanks Luud!

Answered 5 years ago

Dhr. L.S. Petitiaux

**Paul G. Macon** · Answer 4 · 2019-05-15T14:42:25+00:00

2

Thank you for your suggestion Luud! I will try that.

Answered 5 years ago

Paul G. Macon

**Luud Roorda** · Answer 5 · 2019-05-14T14:42:37+00:00

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.