RNA Extraction from Listeria monocytogenes Harvested from Infected Murine Macrophages

Take a tube with filter paper containing Listeria monocytogenes. These bacteria are harvested from infected murine macrophages.

Add a buffer containing RNase inhibitors to prevent RNA degradation, then vortex the tube at multiple angles to dislodge the bacteria.

Centrifuge and transfer the suspension to a tube containing a phenol–chloroform–detergent mixture.

Vortex and incubate at an elevated temperature to disrupt bacterial membranes, inactivate proteins, and lyse the bacteria, releasing intracellular components.

Centrifuge to separate the RNA-rich aqueous phase from the other components.

Collect the RNA and transfer it to a tube containing sodium acetate and ethanol.

Vortex, then incubate at a low temperature.

Sodium acetate neutralizes the RNA’s negative charge, while ethanol and cold conditions promote RNA precipitation.

Centrifuge to pellet the RNA.

Remove the supernatant and wash the pellet with cold ethanol.

Centrifuge again, remove the supernatant, air-dry, and add RNase-free water.

Store the RNA for further analysis.

Thaw the tubes containing the filters on ice to keep them cold. Then to each tube, add 650 microliters of cold acetate EDTA or AE buffer.

Working as quickly as possible,