Hi everyone,

I am currently working on RNA extraction from bacterial (gram-neg) cultures for whole-transcritpome sequencing.

I use a protocol as follow:

Bacterial cell pellets are resuspended in CTAB lysis buffer and 20 % SDS and 10 % N-Lauroylsarcosin are added. The cells are mechanically broken using a Homogenizer and glass beads.

The cell suspension is treated with Lysozyme and digested using Proteinase K.

Following to this, a 25:24:1 mixture of Phenol:Chloroform:Isoamylalcohol is added, properly mixed and centrifuged at high speed at 4°C for 10 min.

The resulting phase seperation I get is not satisfing. In the first trials the interphase was crowded with bulky, yellowish protein particels and I could only take a small amount from the top. For the next experiments, I was splitting the cell culture before centrifugation in order to get less cell material and proceeded in the same way. For this, the phase separation was even worse, the obtained interphase was slippery and white clouds started to dissolve into the liquid layer stright after removing the tubes from the centrifuge. It was impossible to take a proper, clean aquaeous phase from this and impossible to continue working with this

I already tried different speeds of centrifugation, enzymes and even tubes but non of this could solve my problems.

However, when proceeding with the material from the bukly interphase samples I do get a nice amount of RNA. RNA is precipitated using PEG6000/NaCl and addtion of gylcogen. Samples are DNase treated on the next day after the extraction.

Even though am still confused, and can not explain where this issues come from and why no "proper" interphase is formed. This is not similiar to any RNA extraction I performed when using Phenol-Chloroform.

I am happy to share experience if someone is or was using similiar protocols or recommendations of how to improve.

Cheers,

Franziska