I want to extract the total bacterial RNA directly from sample collected in PBS. I cant enrich the bacteria as my research focuses on direct isolation of rna. As bacterial count is less, i also need to filter out the bacteria.
I dont have any bead-beater in my lab. So, I cannot use MOBIO Powerwater kit to isolate rna from bacteria filtered with .22 um membrane filter.
I have TRIzol available in my lab. So I want to follow the steps below. Please tell me if it is okay or suggest modifications what I can do with TRIzol reagent.
Modified Homogenization steps by using TRIzol-
1. separate the bacteria with .22 um membrane filter (1 cm in diameter).
2. put the membrane filter in a microfuge tube.
3. add 750 ul TRIzol reagent inside the tube.
4. crash / homogenize the membrane filter with IQ plus tissue homogenizer.
5. centrifuge for 2 min at 13000 rpm. (???? need more duration??)
6. collect the supernatant from the tube and proceed to the subsequent manufacturer protocol of TRIzol reagent.
Thanks in advance.