This article details a protocol for isolating RNA from Listeria monocytogenes harvested from infected murine macrophages. The method involves multiple steps including vortexing, centrifugation, and precipitation to ensure high-quality RNA extraction.
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, vigorously vortex the filter tube so that the filter whisks to the periphery of the tube and the buffer fully washes the filter. It may be necessary to invert the tube and vortex to fully wash off the bacteria from the filter. The most critical step of the procedure is to ensure the bacteria are efficiently removed from the filter.
To do so, we repeat vortexing the tubes multiple times in different positions. Once the rest of the samples have been vortexed, briefly spin down the tubes. Next, after transferring the bacteria containing buffer from each tube to the tubes of SDS and phenol chloroform, place the tubes in a multi-tube vortex device and vortex at full speed for 10 minutes.
Then, incubate the tubes in a heat block at 65 degrees Celsius for 10 minutes before spinning at maximum speed for five minutes. Transfer the aqueous layer to a fresh tube containing 40 microliters of three molar sodium acetate and one milliliter of 100% ethanol. After thoroughly vortexing the samples, incubate the tubes at minus 80 degrees Celsius for one hour or at minus 20 degrees Celsius overnight.
Then, after centrifuging the samples, carefully aspirate ethanol from each tube before adding 500 microliters of cold 70% ethanol to each sample. After vortexing and spinning the samples again, carefully aspirate the ethanol from each tube and use a vacuum evaporator to dry the pellets for two minutes. Next, add 25 microliters of RNAse free water to each tube, and incubate at room temperature for 20 minutes.
繁體中文
