A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.
Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).
1. LNA probes & experimental design
Solutions
2. Fixation
3. Diethyl pyrocarbonate treatment
4. Permeabilization
5. Hybridization
6. Post-hybridization washes
7. Blocking and staining
8. Representative results
An example of cells that are fixed and permeabilized effectively is shown in the flow cytometry dot plot in Figure 3A. When using the conditions described above for permeabilization, the cell population is small and homogenous indicating few cell aggregates. When higher (5 mg/mL) concentrations of lysozyme are used, cells are more likely to aggregate and show increased forward scatter values, indicative of larger particles (Figure 3B). An example of flow cytometry data from a successful LNA flow-FISH experiment is shown in Figure 4. The histogram demonstrates the specific detection of a target sRNA along with three negative controls. The ‘no dye’ negative control produces the least fluorescence, followed by the ‘no LNA’ and ‘non-expressed sRNA’ negative controls. Finally, the sample with the sRNA-specific LNA probe produces the greatest fluorescence. Once the method and LNA probes are validated in this manner, additional experiments can be designed to monitor changes in the sRNA signal over time, in response to mutations or varied culture conditions, etc.
Figure 1. Depiction of the overall scheme of a LNA flow-FISH experiment for the detection of bacterial sRNA.
Figure 2. Staining and amplification of the LNA flow-FISH signal. a) Hybridization of the biotinylated LNA probe. b) Staining with the fluorescent DyLight 488 streptavidin conjugate. c) Binding of the biotinylated anti-streptavidin antibody to the DyLight 488 streptavidin. The antibody can bind streptavidin through its antigen-binding site or can be bound by streptavidin through its biotin residues. d) Amplification of the signal by further staining with the fluorescent DyLight 488 streptavidin conjugate.
Figure 3. Flow cytometry dot plots showing forward versus side scatter results for a) 1 mg/mL lysozyme, 3 μg/mL proteinase K permeabilization and b) 5 mg/mL lysozyme, 3 μg/ml proteinase K permeabilization.
Figure 4. Histogram analysis of the LNA flow-FISH results. The fluorescent signal generated from each sample is shown by the four traces: black – sRNA-specific LNA probe; red – ‘non-expressed sRNA’ negative control; blue – ‘no LNA’ negative control; purple – ‘no dye’ negative control.
The LNA flow-FISH method presented here was used to detect the expression of a sRNA from the Gram-negative marine bacterium Vibrio campbellii whose expression has previously been confirmed via microarray-based expression profiling and reverse transcription polymerase chain reaction16. To date, we have used this method to monitor the expression of a variety of RNAs (e.g. trans-encoded sRNA, sRNA that modulate protein activity, riboswitches, and mRNA). As such, we are confident that the method is adaptable and can be used to detect any sRNA or mRNA target and can be modified for use in any bacterial species or cell type. If modification of this protocol is necessary for other cell types, the most important variable to consider manipulating is the permeabilization step. For example, when modifying the permeabilization conditions described here, changes in the concentrations of lysozyme and proteinase K should be tested. We found that doubling or tripling the amount of lysozyme and proteinase K resulted in major changes in the LNA flow-FISH results. Furthermore, when testing additional permeabilization conditions, the cells should be analyzed for clumping, cell loss, and the best obtainable signal to background fluorescence ratio. The extent of cell clumping can be tested for by microscopy and/or flow cytometry. By flow cytometry, cell clumps are present as tails in a forward vs. side scatter dot plot and the correct permeabilization method will minimize this tailing effect. This method is also amenable to multiplex sRNA detection without major modifications to the described protocol as additional LNA probes labeled with other haptens such as digoxigenin could be added during the hybridization step and then detected with an anti-digoxigenin antibody and secondary antibody-fluorophore conjugate. Currently, the only limitation that we have encountered with this method is its inability to generate sufficient signal from weakly expressed sRNA species and efforts are underway to determine the detection threshold (in absolute copy number per cell).
Overall, the LNA flow-FISH method provides the opportunity to measure bacterial sRNA or mRNA expression at the single cell-level in a high throughput manner to provide insights on the presence and relative abundance of an sRNA species and the degree to which its expression varies in a population.
The authors have nothing to disclose.
This work was supported by the Office of Naval Research via US Naval Research Laboratory core funds.
Name of the reagent | Company | Catalogue number | コメント |
10X Phosphate buffered saline (PBS), pH 7.4 | Applied Biosystems/Ambion | AM9625 | |
Nuclease-free water | Applied Biosystems/Ambion | AM9932 | (not DEPC treated) |
32% paraformaldehyde | Electron Microscopy Sciences | RT 15714 | Ethanol free |
Acetic acid | Acros Organics | 327290010 | |
Diethyl pyrocarbonate (DEPC) | Sigma Aldrich | D5758 | Keep desiccated |
Lysozyme | Sigma Aldrich | L2879 | |
Proteinase K (20 mg/mL) | Applied Biosystems/Ambion | AM2546 | |
Formamide | Applied Biosystems/Ambion | AM9342 | Deionized, aliquot and freeze |
Dextran sulfate MW > 500,000 | Sigma Aldrich | D8906 | Aliquot 60% solution and freeze |
Denhardt’s solution 50X concentrate | Sigma Aldrich | D2532 | Aliquot and freeze |
Sodium citrate buffer 20X | Applied Biosystems/Ambion | AM9770 | |
Salmon sperm DNA (sheared) 10 mg/mL | Applied Biosystems/Ambion | AM9680 | Aliquot and freeze |
Yeast tRNA (10 mg/mL) | Life Technologies/Invitrogen | 15401-011 | Aliquot and freeze |
Tween-20 | Sigma Aldrich | P9416 | |
5X in situ hybridization blocking solution | Vector Laboratories | MB-1220 | |
DyLight 488 streptavidin | Vector Laboratories | SA-5488-1 | |
Biotinylated anti-streptavidin | Vector Laboratories | BA-0500 | Aliquot and freeze |