RNA Extraction from Listeria monocytogenes Harvested from Infected Murine Macrophages

Abstract

Source: Sigal, N., et al. RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells. J. Vis. Exp. (2016)

This video demonstrates RNA extraction from Listeria monocytogenes isolated from infected murine macrophages. The protocol preserves RNA, lyses cells using phenol–chloroform and heat, and separates RNA by centrifugation. Salt and ethanol, combined with cold incubation, promote RNA precipitation. The resulting RNA captures host-induced gene expression, revealing bacterial virulence and adaptation strategies.

Protocol

1. Nucleic Acids Extraction

NOTE: Perform all manipulations with phenol and chloroform solutions in a fume hood.

1. Prepare a 1:1 mix of acidified phenol: chloroform, 400 µl for each sample. Mix in separate tubes, and then withdraw the resulting aqueous layer by aspiration. Add 40 µl of 10% SDS.
2. Thaw the filter-containing tubes on ice to keep them cold. To each filter-containing tube add 650 µl of acetate-EDTA (AE) buffer (50 mM NaOAc pH 5.2, 10 mM EDTA, RNase free water).
3. Perform the following steps as quickly as possible.
   1. Vigorously vortex the tube containing the filter so that the filter whisks to the periphery of the tube and the buffer fully washes the filter. Always keep the tube cold by placing it back on ice.
      NOTE: It may be necessary to additionally vortex the tube while inverted to fully wash the bacteria off the filter.
   2. Repeat for all tubes. It may be useful to spin-down the suspension shortly (1 min at 120 x g) at the end of the process.
4. Transfer the bacteria-containing buffer to a 1.5 ml microcentrifuge tube containing the SDS and phenol/chloroform mix. Repeat for all tubes. It may be necessary to spin the filter containing tubes (1 min at 120 x g) again to get residual buffer from the filter.
5. Place all the 1.5 ml tubes in a multi-tube vortex device, and vortex at full speed for 10 min.
6. Incubate the tubes in a 65 °C heat block for 10 min. Centrifuge at maximum speed (> 14,000 x g) for 5 min.
7. Transfer the aqueous layer (about 400 µl) from each tube to a new 1.5 ml tube containing 40 µl of 3 M sodium acetate (pH 5.2) and 1.0 ml of 100% ethanol. Vortex each tube thoroughly.
   NOTE: Glycogen can be added at this step to improve visualization of the precipitated pellet.
8. Incubate the samples at -80 °C for 1 hr. Alternatively, incubate samples at -20 °C overnight. Then, centrifuge the samples at 4 °C for 20 min at maximum speed (> 14,000 x g).
9. Carefully aspirate off the ethanol (upper layer) from each tube. Add 500 µl cold 70% ethanol to each sample and vortex thoroughly. Centrifuge at 4 °C for 20 min at maximum speed (> 1 4,000 x g).
10. Carefully aspirate off the ethanol from each tube. Dry the samples for approximately 2 min using a vacuum evaporator with no heat. Do not over-dry the samples.
11. Add 25 µl RNase-free water to each sample. Incubate at room temperature for 20 min. Carefully vortex and spin-down.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Listeria monocytogenes 10403S
H2O, RNAse free	Thermo Scientific	10977-015	DEPC-treated water can be used
Phenol saturated pH 4.3	Fisher	BP1751I-400
Chloroform	Fisher	BP1145-1
Iso-amyl alcohol	Sigma-Aldrich	W205702
Sodium acetate	Sigma-Aldrich	W302406
EDTA	Sigma-Aldrich	EDS
SDS 10%	Sigma-Aldrich	L4522
Ethanol absolute	Merck Millipore	1070174000
MF-Millipore filters 45 mm, 0.45 µm	Merck Millipore	HAWP04700
Vortex-Genie 2	Scientific Industries	Model G560E
65 °C heat block	Thermo Scientific
4 °C table centrifuge	Eppendorf	5417R
Sterile pipettes, 25 ml	Greiner
Falcon tubes, 50 ml	Greiner
1.7 ml tubes, RNase-free	Axygen	MCT-175-C
SpeedVac system	Thermo Scientific	SPD131DDA