Hello Aayush Bahl

Since mycobacteria have thick cell walls, total RNA can be extracted without considerable degradation if a rapid cell lysis protocol is synchronized with the RNA extraction protocol. In the absence of bead beater and with no success with lysozyme, you may use ultrasonic treatment as an effective method to lyse the cells. You may mix the cells with TRIzol and sonicate for different time points to release intact RNA from the cells.

Protocol:

1. To the cell pellet add TRIzol reagent and sonicate the cell suspension at the optimized time point followed by three to four cycles of freeze-thaw.

2. Cell debris may be removed by centrifugation at 5000 rpm for 2 mins.

3. The supernatant fluid is extracted with 750ul of chloroform.

4. To the aqueous phase add 10ul of 100ug/ml RNase free glycogen as a carrier.

5. Precipitate the RNA by adding 0.6 volumes of isopropanol and incubate for 10 mins at room temperature.

6. RNA pellet is recovered by centrifuging at 15000 rpm for 15 mins at 4 deg C.

7. Wash the pellet once with 70% ethanol.

8. Air-dry and resuspend the pellet in 40ul of RNase-free DEPC treated water.

Please note: If desired, the RNA sample may be treated with DNase followed by chloroform extraction. The DNase treatment will remove the contaminating DNA, if any, present in the RNA preparation.

You may want to refer to the article attached below.

Article A rapid protocol for isolation of RNA from mycobacteria

Best.

Thank you so much Malcolm Nobre !

in my experience isopropanol precipitation of RNA helps at -20 overnight

Julie Ann Dougherty Thank you so much, this really helped!