The overall goal of this procedure is to detect the expression of bacterial RNA in individual bacterial cells. This is accomplished first by designing a locked nucleic acid or LNA probe with a biotin TEG modification on the five prime end. If the bacterial cells are then fixed, treated with dathyl, percarbonate or dsy and then permeated next, the RNA is denatured and the LNA probe is hybridized.
Third, the cells are washed, blocked, and stained. Finally, the cellular fluorescence is measured by flow cytometry. Ultimately changes in RNA expression over time and within populations can be evaluated.
This method can help answer key questions regarding the relative presence and abundance of RNA species within a single cell, and the degree to which this expression changes within homogeneous or heterogeneous populations. Though this method is currently being used to monitor bacterial small RNA expression, in theory, it can be used to detect RNA in potentially any cell type. After designing the LNA probe sequence, use blast to ensure that the probe is specific only to the SRNA or mRNA of interest.
When ordering the probe, add a biotin TEG modification to the five prime end of the LNA oligonucleotide. The biotin elation is necessary for post hybridization staining with a stripped avid and di conjugate. Three negative controls should be included in the method one A, no LNA control in which an LNA probe is not added during the hybridization.
Step two, A no die control in which the LNA probe is hybridized to the target SRNA, but the hybridization event is not detected due to the absence of the fluorescent stain. And three, A non expressed SRNA or mRNA control that utilizes an LNA probe that targets a non-existent or non expressed SRNA in order to monitor non-specific hybridization begin by harvesting one times 10 to the eighth cells of the bacteria of interest and transferring them into a 1.5 milliliter micro centrifuge tube. Then pellet the bacterial cells at 2300 times G for five minutes after centrifuging.
Remove the supernatant by pipetting and not by decanting. Next, resuspend the pellet in 400 microliters of PBS add 400 microliters of A PFA acetic acid PBS fixative solution and mix the cells well. Now incubate the suspension for 10 minutes at 25 degrees Celsius.
Vortexing once after five minutes after spinning down the cells again. This time at 4, 500 times G for two minutes. Wash the pellet two times with 400 microliters of PBS.
Finally resuspend the pellet in 400 microliters of PBS and store the now fixed cells at four degrees for up to seven days. Begin by adding 400 microliters of a freshly prepared point, 1%dsy solution to each fixed bacterial cell sample. And then mixing the cell solutions well by pipetting.
Incubate this mixture for 12 minutes, a 25 degrees Celsius, and then after washing the cells twice with 400 microliters of PBS add 800 microliters of a freshly prepared lysozyme solution to the cells. Mix the cell lysozyme solution thoroughly by vortexing and incubate the cells for 30 minutes at 25 degrees Celsius with occasional mixing. After spinning down the cells, add 800 microliters of a freshly prepared proteinase K solution to the pellet and mix the resuspended cell solution thoroughly by pipetting.
Incubate the cells for 15 minutes at 25 degrees Celsius vortexing halfway through the incubation period after spinning down and washing the cells with 400 microliters of PBS resuspend the cells in another 400 microliters of PBS and add 100 microliters of the suspension into four separate 1.5 milliliter micro centrifuge tubes, then palate the cells and discard the supernatants. Now incubate all four samples in 100 microliters each of hybridization buffer at 60 degrees Celsius for 60 minutes. Following the hybridization, add one milliliter of SSC plus tween 20 or SSCT to the hybridization mixture to allow the cells to be removed from the viscous hybridization solution.
After pelleting, the cells add 200 microliters of form amide SSCT solution to the pellets and mix thoroughly. Then incubate the samples for 30 minutes at 65 degrees Celsius in a heating block. Now add one milliliter of SSCT to the mixture and after pelleting the cells once again, resus suspend the cells in 500 microliters of SSCT and incubate the cell solution for 40 minutes on the heat block.
After pelleting, the hybridized cells add 200 microliters the blocking buffer to the pellets, and then incubate the cells for 30 minutes at 25 degrees Celsius. After pelleting, the cells again add 50 microliters of a stripped avid and dye solution to the pellets and mix the cells thoroughly for the no dye control. Add 50 microliters of blocking buffer.
Only incubate all of the samples for another 12 minutes at 25 degrees Celsius with constant mixing in a thermo mixer, protecting the tubes from light using aluminum foil. Next, wash the cells once with 500 microliters of SSCT buffer and once with 400 microliters of PBS plus tween 20 or PBST. After removing the supernatants add 100 microliters of biotinylated antis, strippin and PBS for 30 minutes at 25 degrees Celsius with occasional mixing, this antibody will bind to the strippin DI conjugate introduced in the previous step.
Then after pelleting and washing the cells twice with 400 microliters of PBST stain a second time by adding 50 microliters of the stripped avid and dye solution to the cells and mixed thoroughly. This step increases the signal output per hybridized LNA probe for the no dye control. Again, add 50 microliters of blocking buffer only, and then incubate all of the tubes for 12 minutes at 25 degrees Celsius with constant mixing in a thermo mixer.
After incubation with the dye solution, wash the cells once again with 500 microliters of SSCT buffer and once with 400 microliters of PBST after the second wash. Resus suspend the pellet in 200 microliters of PBST and store the cells at four degrees Celsius until ready for flow cytometry analysis for smaller bacterial cells set the flow rate on the flow cytometer to slow to decrease the core size. Additionally for flow cytometers with forward scatter threshold cutoffs, set the cutoff as low as possible to ensure that the small bacterial cells are detected.
An example of cells that are fixed and perme effectively is shown here in this flow cytometry dot plot when using the conditions just described. For permeation, the cell population is small and homogenous, indicating few cell aggregates. When higher concentrations of lysozyme are used, however, cells are more likely to aggregate and show increased forward scatter values indicative of larger particles as seen in this dot plot.
Here an example of flow cytometry data from a successful LNA flow fish experiment is shown. The histogram demonstrates the specific detection of a target SRNA along with three negative controls. The no die negative control in purple produces the least fluorescence, followed by the no LNA and non expressed SRNA negative controls in blue and red respectively.
Finally, the sample with the SRNA specific LNA probe produces the greatest fluorescence in black. Once mastered, this technique can be performed in approximately six hours if performed correctly. In addition, this technique provides more information and is more cost effective than R-T-P-C-R.
Following this procedure, additional probes can be labeled with other HAPS and added during the hybridization step to enable multiplex RNA detection.I.
