The overall goal of this procedure is to extract RNA from bacteria grown intracellularly in macrophage cells. This method can help answer key questions in the bacterial pathogenesis field, such as, what are the factors in genes that bacteria trigger in order to mediate infection? The main advantage of this technique is that it significantly enriches for bacterial RNA while minimizing contamination of host nucleic acids.
Demonstrating the procedure will be Anna Pasechneck, a PhD student from my laboratory. To prepare cells for bacterial infection, on day one, use 145 millimeter dishes to seed two times ten to the seven bone marrow derived macrophage, or BMDM cells, in 30 milliliters of BMDM medium plus Pen/Strep. Incubate at 37 degrees Celsius and five percent carbon dioxide in a forced air incubator overnight.
Inoculate a bacterial culture of wild type L.monocytogenes bacteria in 10 milliliters of brain heart infusion, or BHI, medium. Place tubes in an incubator in a slanted position and incubate at 30 degrees Celsius without shaking overnight. The following day, pre-warm BMDM medium without antibiotics and PBS to 37 degrees Celsius.
Then use 25 milliliters of the warm PBS to wash the macrophage monolayer twice to remove the antibiotics before adding 30 milliliters of fresh BMDM medium to the cells. Next, wash the overnight L.monocytogenes culture by centrifuging 1.5 milliliters of the bacteria. Then use 1.5 milliliters of PBS to re-suspend the pellet.
Then, using 0.5 milliliters of the washed bacterial suspension, infect each macrophage plate. If using multiple plates, space the infections 15 minutes apart to allow for harvesting each plate individually at the end of the infection. Following a 30 minutes incubation at 37 degrees Celsius, use PBS to wash the infected cells twice to remove unattached bacteria, and then add 30 milliliters of pre-warmed BMDM medium.
Incubate for 30 minutes to allow bacteria to internalize. At one hour post infection, add gentamicin to a final concentration of 50 micrograms per milliliter to kill any remaining extracellular bacteria. Assemble the filter apparatus by placing a filter head on a collecting liquid flask with a vacuum outlet port.
Then place a 0.45 micron filter on the filter head, followed by a cylinder funnel, and use a metal clamp to secure the different parts. At six hours post infection, examine the cell monolayer under the microscope before harvesting. Working with one plate at a time to harvest the bacteria, use PBS to wash the infected cells.
Then, to lyse the macrophage cells, add 20 milliliters of ice cold RNAse free water and quickly but carefully use a cell scraper to scrape the cells off the plate. After collecting the cells into a 50 milliliter conical tube, vortex for 30 seconds, then centrifuge at 800 times g and four degrees Celsius for three minutes to remove macrophage cell nuclei. To collect bacteria, pass the supernatant through the filter using the vacuum system.
Then, with tweezers, roll the filter and quickly transfer it to a 15 milliliter conical tube. Immediately use liquid nitrogen to snap freeze the tube. On the third day, prepare 400 microliters of a one to one mixture of acidified phenol and chloroform into separate 1.5 milliliter tubes for each sample.
After mixing well, aspirate the top aqueous layer before adding 40 microliters of 10%SDS to the remaining solution. 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.
After vortexing and spinning down the samples, with a micro-volume UV/VIS spectrophotometer, measure the RNA concentration. Expect to extract about 0.5 to one microgram of nucleic acids per plate. To carry out Dnase treatment, set up the reactions in microcentrifuge tubes according to the text protocol.
Incubate the samples at 37 degrees Celsius for 45 minutes. Then add 450 microliters of RNAse free water and 500 microliters of phenol chloroform IAA mix. Once the samples have been vortexed and spun at maximum speed for two minutes, transfer the aqueous layer to a new tube and add 500 microliters of chloroform IAA mix.
After vortexing and spinning the samples again, transfer the aqueous layer to a new tube, before adding one milliliter of ethanol and 15 microliters of three molar sodium acetate. Following another vortex, incubate the tubes at minus 80 degrees Celsius for one hour or minus 20 degrees Celsius overnight. After the incubation, centrifuge at maximum speed and four degrees Celsius for 20 minutes.
Carefully aspirate off the ethanol from each tube. Then add 500 microliters of cold 70%ethanol to each sample. After vortexing and spinning as before, carefully aspirate the ethanol from the tubes and use an unheated vacuum evaporator to dry the samples for two minutes without over drying.
Then, add 12 microliters of RNAse free water, incubate at room temperature for two minutes, and vortex the samples before spinning them down. Immediately place the purified RNA samples on ice. Finally, use a micro-volume UV/VIS spectrophotometer to measure the RNA concentrations.
Expect approximately 100 nanograms of RNA per combined samples. The Listeria RNA purified from macrophage cells is suitable for downstream transcription analysis by multiple available techniques. Shown here are the results of an RT qPCR experiment used to validate the transcription of known virulence genes during wild type L.monocytogenes growth in macrophages in comparison to exponential growth in the rich laboratory medium, BHI.
In particular, the results of the hly and actA genes at six hours post infection are illustrated. Transcription levels were normalized to the levels of 16S ribosomal RNA as a reference gene. The data shown are representative of three independent biological repeats.
Once mastered, this technique yields about 100 nanogram of total RNA, which is suitable for transcriptomic analysis by different methods, such as RT qPCR, microarray, and RNA-seq. While attempting this procedure, it is important to remember to work quickly as possible to avoid changes in gene transcription. While this protocol describes the purification of Listeria RNA, it can be easily modified to other bacterial pathogens.
