This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.
A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).
Stochasticity is a fundamental and unavoidable aspect of gene expression and gives rise to cell-cell heterogeneity1, both at the level of transcripts and proteins2,3. Quantifying the variability between cells under well-defined conditions offers a unique window into the basic processes that underlie gene expression and its regulation. One important source of cell-cell heterogeneity in bacteria takes place at the transcriptional level. Transcript numbers vary not only due to the stochasticity of transcription, but also to post-transcriptional processes such as regulation by small RNAs and RNAases2. One way of directly accessing this heterogeneity in a quantitative fashion is by fluorescently tagging individual transcripts of a given gene in smFISH. This methodology allows the detection and subcellular localization of particular RNA molecules in fixed, individual bacterial cells4. mRNAs are hybridized with a set of fluorescently-labeled ~20 base-long oligonucleotides that are designed to bind selectively to transcripts of interest5,6. Multiple labeling ensures detection above background fluorescence, and individual mRNA molecules appear as diffraction-limited spots under a fluorescence microscope7 (see Figure 1). There are other approaches for labeling mRNA molecules, in which the complementary oligomer probes carry conjugated haptens (e.g., biotin or digoxigenin) that are detected using secondary fluorescently-labeled reporter techniques8.
There are other methods that provide quantitative information about transcripts, in addition to smFISH. Some, such as the Northern blot or quantitative PCR, probe the bulk and thus can measure neither the number of mRNA copies nor their position in individual cells. Therefore these methods are not suitable to quantify cell-to-cell variability. A recent image-based technique that allows for the quantification of both the copy number of RNAs within cells as well as their intracellular location, called multiplexed error-robust fluorescence in situ hybridization (MERFISH) has been developed. MERFISH is based on the assignment of a unique barcode consisting of a defined combination from a fixed number of fluorescently-labeled oligonucleotide probes. These barcodes are read out in sequential rounds of smFISH measurements, with photobleaching following each round of hybridization, thereby increasing throughput by two orders of magnitude9,10. This technique necessitates an automated fluid handling system and the proper design of the probe set.
The combination of multiple fluorescence labeling of individual transcripts, together with novel super-resolution techniques such as stochastic optical reconstruction microscopy (STORM)11, enables a ten-fold increase in resolution in the subcellular localization of transcripts. In STORM, a suitable combination of fluorescent probes and imaging buffer allows for multiple cycles of fluorescence emission per probe molecule (blinking). STORM may also be used to image the E. coli transcriptome and observe genome-wide spatial organization of RNA, by labeling simultaneously all the transcripts of interest12.
All the single-cell methods reviewed above are based on imaging transcripts in fixed cells. Hence, they do not provide any information regarding the kinetic properties of transcripts within cells. To follow transcripts in live cells13, mRNAs can be labeled by the fusion of the gene of interest to an array of binding sites. These latter are then recognized by an RNA-binding protein, such as the bacteriophage MS2 coat protein, which is fused to a fluorescent protein such as the green fluorescent protein (GFP)10,14,15.
Here we describe a method for labeling individual mRNAs with a set of fluorescently-labeled DNA probes, for use in smFISH experiments, in particular in E. coli. Furthermore, we show that the same labeling scheme may be used for STORM measurements with minor modifications.
1. Probe Design
NOTE: This protocol uses commercially available oligonucleotide probes already tagged with fluorophores. The probes consist of a set of specific sequences complementary to a target mRNA, each probe being conjugated to a single fluorescent molecule. Alternatively, it is possible to attach fluorescent markers to probes, as described elsewhere5,16.
2. Reagent Preparation
NOTE: It is important to keep the samples RNAse-free by using RNase-free consumables such as filtered pipette tips and tubes, and to keep any working environment clean. In order to avoid degradation of the target transcripts, all reagents used following cell fixation must be nuclease-free (see table of materials) or diethylpyrocarbonate (DEPC)-treated and sterilized.
3. Sample Fixation
4. Sample Permeabilization
5. Hybridization
6. Washing
7. Preparation of Samples for Imaging
NOTE: Cells need to be immobilized in order to allow proper imaging under a fluorescence and phase-contrast microscope. The following methods can be used to prepare samples in which different fields of view can be imaged at different focal planes.
8. Transcript Visualization
9. Data Analysis
We carried out smFISH measurements of galK and sodB transcripts in E. coli cells. The transcripts were hybridized with a set of specific sequences complementary to the target sequence, each probe being conjugated to a single fluorescent molecule (see table of materials). Fluorescence and phase contrast images of MG1655 wild-type E. coli strain (WT) or a JW0740 (Keio collection)19 galK-deleted strain (ΔgalK) were exposed to 2 mM D-fucose are shown in Figure 1A and 1B. Cells were fixed at least 3 h after exposure to D-fucose. The panels in Figure 1A (top) show one frame out of 13, corresponding to the height at which cell contours are in focus, representing the level of galK induction in response to variable extracellular D-fucose concentrations. Spots within cells in the fluorescence images correspond to either single or few galk transcripts, and the determination of the transcript number in each cell is carried out by quantifying localized fluorescence. Numerous spots are seen in most cells, when 2 mM D-fucose is added to the bacterial culture, whereas a few spots are seen in the absence of extracellular D-fucose. In the ΔgalK strain no spots are observed over the background as anticipated. Surface labeling with lipophilic fluorescent marker is shown in Figure 1B (bottom).
In addition, we imaged the sodB transcripts in E. coli bacterial culture grown in LB medium in the logarithmic growth phase. Fluorescence and phase contrast images of smFISH in WT or a JW1648 (Keio collection)19 sodB-deleted strain (ΔsodB) are shown in Figure 2A. The panels in Figure 2A (top) show one frame out of 13, corresponding to the height at which cell contours are in focus, representing the level of sodB. In the ΔsodB strain, no spots are observed over the background, as anticipated.
The same cultures were imaged by STORM (top) and the perimeters of the cells were determined with surface labeling using a lipophilic fluorescent marker to illustrate the localization of sodB within the cells. Staining the membrane allows accurate positioning of transcripts in the cell (Figure 2B). The ΔsodB strain was taken as the background signal and to determine the minimal cluster size (see Figure 2B). The mean transcript number of smFISH2 and STORM is comparable.
Figure 1: Effects of D-fucose on galK transcripts visualized using smFISH. (A) Top: smFISH images of fluorescence of galK mRNA in individual E. coli cells. An MG1655 wild-type strain (WT) or a JW0740 (Keio collection) galK-deleted strain (ΔgalK)19 were grown with or without 2 mM D-fucose. The galK transcripts were hybridized to complementary probes conjugated with a dye optically equivalent to Cy3 (see table of materials). Bottom: same fields of view in phase-contrast. (B) WT cells were grown with or without 2 mM D-fucose as above and labeled with 2 μM of a lipophilic membrane dye (see table of materials) for 20 min prior to fixation. Top: fluorescence images of galK mRNA using smFISH. Images were taken with a microscope controlled by a commercial software using a 100× N.A 1.45 oil immersion phase contrast objective lens (see table of materials) and an EMCCD camera (see table of materials). The filters used were as described in the table of materials. A phase contrast image was acquired followed by a z-stack of 13 slices and 250 nm spacing of fluorescent images with 2 s integration time for each slice. Middle: same fields of view with phase-contrast imaging. Bottom: cells labeled with lipophilic fluorescent markers, with appropriate filters described in the Materials Table. Please click here to view a larger version of this figure.
Figure 2: Imaging of sodB transcripts labeled with the same probe set in smFISH and STORM. (A) Top: smFISH images of fluorescence of sodB mRNA in individual E. coli cells. An MG1655 wild-type strain (WT) or a JW1648 (Keio collection) sodB-deleted strain (ΔsodB)19 were grown in LB medium. The sodB transcripts were hybridized to complementary probes conjugated with a dye optically equivalent to Cy5 (see table of materials). Bottom: same fields of view in phase-contrast. (B) Overlay of fluorescent molecules (individual colored dots) localized by STORM (670 nm emission) and a snapshot of cell membranes in the same field of view. The cells were labeled with a lipophilic dye, excited with an argon laser at 488 nm at 1% of maximal power (0.05 kW/cm2) with an emission peak at 510 nm and imaged with the appropriate filter (see table of materials). WT and sodB cells were grown as described above and labeled with a 2 μM lipophilic membrane dye (see table of materials) for 20 min prior to fixation. Super-resolution images were recorded with a commercial microscope (see table of materials). Transcripts labelled with a dye optically equivalent to Cy5 were localized using an excitation laser at 647 nm in an imaging buffer for STORM. Images were recorded using a 60x, NA 1.2 water immersion objective (see table of materials) and an EMCCD camera (see table of materials) with gain set at 50, frame rate at 50 Hz, and maximal power of 647 and 405 nm lasers set at 5 and 0.05 kW/cm2, respectively. The total number of frames acquired was 8,000. Data was analysed using commercial software (see Materials Table). Please click here to view a larger version of this figure.
We have measured in our laboratory the transcript number of different genes in E. coli cells using the smFISH method2. In brief, this procedure consists of the following steps: cell fixation, permeabilization of membranes to allow for probe penetration, probe hybridization, and sample imaging using a standard fluorescence microscope. This procedure is based on previously published ones with some modifications6,7,16. It has been previously reported that smFISH requires that the number of oligonucleotide probes lie in the range 48-72, in order to achieve a signal lying well above the background7. We have shown that this number may be actually smaller20, depending on the gene sequence of interest, the optical setup, and the specific experimental conditions.
Each probe should be 17-22 nucleotides long, with an inter-probe separation of at least two nucleotides and a GC content of ~45%, in order to reduce the effects of non-specific, off-target binding. Some probes may fail to bind a target, and the overall efficiency of binding can be optimized by modifications of experimental procedures such as the conditions of fixation and hybridization21. An important factor that must be taken into account is the choice of fluorophores. It is highly recommended to label the probes with a dye with low susceptibility to photo-bleaching. Photo-bleaching minimization can be achieved by optimization of exposure times, illumination sources, and integration times of image acquisition, and by the addition of an oxygen scavenging system.
The extent of non-specific binding events should be assessed by carrying out smFISH experiments with cells from strains in which target genes are deleted, for example from the Keio collection19. If the background signal is high, the number and duration of washing steps should be increased, or cells should be incubated in wash buffer at 30 °C for 1 h and then washed twice. Furthermore, to reduce the background, it is suggested to optimize the formamide concentration (10-40% v/v ratio) in the hybridization and the wash buffers. Increasing the formamide concentration reduces nonspecific binding, but may also reduce the binding of probes to the target mRNA. During the hybridization step, it is important to remove the supernatant entirely; a dilute hybridization buffer may lead to decreased labeling efficiency. In addition, target RNAs must be sufficiently long in order to obtain a well-localized spot above background. Recent improvements in signal to-noise ratios and binding specificity through backbone modification of the probes now make it possible to detect shorter RNA fragments, such as eukaryotic microRNAs or bacterial small non-coding RNAs2,10. We recommend that the mean copy number obtained by smFISH should be validated with quantitative PCR7,16.
We have shown in this manuscript that this method can be modified with minor changes to study the localization of transcripts in E. coli with higher optical resolution using stochastic optical reconstruction microscopy (STORM)11.
In summary, smFISH is a versatile method for RNA illumination that allows the direct measurement of the cell-cell variability in transcript number and the localization of target transcripts in the cell, in both eukaryotes and prokaryotes. It provides quantitative information about basic processes of gene expression and can be easily implemented for STORM imaging.
The authors have nothing to disclose.
This work was supported by an Israel Science Foundation grant 514415 (to J.S.) and a BSF-NSF (MCB) grant 2016707 (to J.S.). Support from a Siegfried and Irma Ullman Professorial Chair (to J.S.) is also acknowledged.
Dextran sulfate sodium salt | Sigma-Aldrich | D8906 | |
Pure Ethanol, 99.5%, ACS reagent, absolute | Mallinckrodt Baker – Avantor | 8025.25 | |
Diethylpyrocarbonate (DEPC) | Sigma-Aldrich | D5758 | |
RNase-free 20X SSC | Life Technologies/Ambion | AM9763 | |
RNase-free 10X PBS | Life Technologies/Ambion | AM9625 | |
TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) BioUltra, for molecular biology | Sigma-Aldrich | 93283 | |
nuclease-free water | Thermo Fisher Scientific | 10977035 | |
Formaldehyde solution for molecular biology, 36.5-38% in water | Sigma-Aldrich | F8775 | |
Deionized formamide, nuclease free | Thermo Fisher Scientific/Ambion | AM9342 | |
E. coli tRNA (ribonucleic acid, transfer type xx from escherichia) | Sigma-Aldrich | R1753-500UN | |
UltraPure BSA (50mg/ml) | Thermo Fisher Scientific/Ambion | AM2616 | |
Vanadyl-ribonucleoside complex,VRC, 200 mM | New England Biolab | S1402S | |
Poly-L-Lysine | Sigma | P4707 | |
Cysteamine and oxygen | Sigma-Aldrich | 30070 | |
Glucose Oxidase from Aspergillus niger, Type VII, 50KU | Sigma-Aldrich | G2133 | |
Catalase | Sigma-Aldrich | C40 | |
D-glucose | Sigma-Aldrich | G8270 | |
D-fucose | Sigma-Aldrich | F8150 | |
Vybrant DiO Cell-Labeling Solution | Life Technologies | V2286 | |
Agarose,low melting reagent | Sigma-Aldrich | A9414 | |
Adhesive silicone isolator 24-2mm Dia. X 1.8 mm depth JTR24R-A2-2.0 | Grace Bio-Labs | JTR24R-A2-2.0 666208 | |
poly-D-lysine-coated glass bottom Glass Bottom Culture Dishes | MatTek Corporation | P35GC-1.5-14-C | |
Super life nitrile powder free examination gloves | Supermax | TC-N-9889 | |
Brand sterilization incubator tape | Sigma-Aldrich | BR61750 | |
Microcentrifuge tubes (1.8 ml) | Axygen – Corning Life Sciences | MCT-175C | |
Falcon round-bottom polypropylene tubes (14 ml) | BD Biosciences | 352059 | |
Conical-bottom centrifuge polypropylene tubes (50 ml) | Corning | 430828 | |
Serological pipettes (Corning 5 ml) | Corning Life Sciences | 4051 | |
Serological pipettes (Corning 10 ml) | Corning Life Sciences | 4488 | |
Serological pipettes (Corning 25 ml) | Corning Life Sciences | 4251 | |
Spectrophotometer cuvettes | Sarstedt | 67.742 | |
RNase-free pipette tips 0.2 – 20 μl | FroggaBio | FT20 | |
RNase-free pipette tips 10 – 200 μl | Axigene/corning | TF-200 | |
RNase-free pipette tips 100 – 1000 μl | FroggaBio | FT1000 | |
RNase-free pipette tips 100 – 1000 μl | Sorenson | 14200 | |
Syringe disposile 10 mL needle G-21 | Becton Dickinson, Biosciences | BD-309643 | |
Minisart 0.2 um Syringe Filter | Sartorius | 16534 K | |
Nikon instruments microscope type A immersion oil A, 8cc | Nikon | MXA20233 | |
Microscope slides 76 x26, 3"x1"x1mm | Thermo Fisher Scientific | 421-004ET | |
#0 coverslip slide 24×60 | Thermo Fisher Scientific/Menzel | BNBB024060A0 | |
Orbital shaker | M.R.C | TOU-50 | |
Hot block | M.R.C | ||
Vortex | Fried Electric Company | G-560-E | |
Microcentrifuge | Eppendorf | 5427R | |
Centrifuge | Eppendorf | 5810R | |
Portable Pipet-Aid XP2, Pipette Controller | Drummond Scientific Company | 4-000-501-I | |
OD600 Spectrophotometer for Bacterial Growth Rates DiluPhotometer | Midsci | OD600-10 | |
iXon X3 EMCCD camera | Andor | DU-897E-CS0-#BV | |
Eclipse Ti microscope | Nikon | MEA53100 | |
CFI plan apochromat DM 100X oil objective lambda PH-3 N.A 1.45 WD 0.13 | Nikon | MRD31905 | |
Filter set (TRITC/CY3): EX – ET545/30X; EM – ET620/60M; BS – T570LP | Nikon | 49005 | |
Filter set (CY5): EX – ET640/30X; EM – ET690/50M; BS – T660P | Nikon | 49009 | |
Nis-elements AR auto reaserch software | Nikon | MQS31000 | |
STORM microscope | Vutara | SR-200 | |
NA 1.2 water immersion objective | Olympus | ||
SRX image acquisition and analysis software | Vutara | ||
Evolve 512 EMCCD camera | Photometrics | ||
Stellaris® FISH Probes, Custom Assay with CAL Fluor® Red 590 Dye | Biosearch Technologies Inc | SMF-1083-5 | |
Probe Sequence for galK mRNA: | |||
gtgttttttctttcagactc | |||
tagccaaatgcgttggcaaa | |||
ctgaatggtgtgagtggcag | |||
caccaatcaaattcacgcgg | |||
acgaaaccgtcgttgtagtc | |||
tgataatcaatcgcgcaggg | |||
gtggtgcacaactgatcacg | |||
acgcgaactttacggtcatc | |||
gctgattttcataatcggct | |||
gcatcgagggaaaactcgtc | |||
gttttcatgtgcgacaatgg | |||
cacgccacgaacgtagttag | |||
ttacgcagttgcagatgttt | |||
tccagtgaagcggaagaact | |||
tgctgcaatacggttccgac | |||
gtccagcggcagatgataaa | |||
tgaccgttaagcgcgatttg | |||
agcctacaaactggttttct | |||
gcggaaattagctgatccat | |||
aaggcatgatctttcttgcc | |||
cagtgagcggcaatcgatca | |||
tgggcatggaaactgctttg | |||
gatgatgacgacagccacac | |||
gggtacgtttgaagttactg | |||
gtgttgtattcgctgccaac | |||
ggtttcgcactgttcacgac | |||
tggctgctggaagaaacgcg | |||
ttcaatggtgacatcacgca | |||
catgcgcaacagcgttgaac | |||
ggcgttttcagtcagtatat | |||
atacgtttcaggtcgccttg | |||
tgagactccgccatcaactc | |||
gaaatcatcgcgcatagagg | |||
caatttgcggcacggtgatt | |||
ttgacgatttctaccagagt | |||
acctttgtcgccaatcacag | |||
ggatcagcgcgacgatacag | |||
atattgttcagcgacagctt | |||
gtctctttaatacctgtttt | |||
ctccttgtgatggtttacaa | |||
Stellaris® FISH Probes, Custom Assay with Quaser Fluor® Red 670 Dye | Biosearch Technologies Inc | SMF-1083-5 | |
Probe Sequence for sodB mRNA: | |||
gtgttttttctttcagactc | |||
tagccaaatgcgttggcaaa | |||
ctgaatggtgtgagtggcag | |||
caccaatcaaattcacgcgg | |||
acgaaaccgtcgttgtagtc | |||
tgataatcaatcgcgcaggg | |||
gtggtgcacaactgatcacg | |||
acgcgaactttacggtcatc | |||
gctgattttcataatcggct | |||
gcatcgagggaaaactcgtc | |||
gttttcatgtgcgacaatgg | |||
cacgccacgaacgtagttag | |||
ttacgcagttgcagatgttt | |||
tccagtgaagcggaagaact | |||
tgctgcaatacggttccgac | |||
gtccagcggcagatgataaa | |||
tgaccgttaagcgcgatttg | |||
agcctacaaactggttttct | |||
gcggaaattagctgatccat | |||
aaggcatgatctttcttgcc | |||
cagtgagcggcaatcgatca | |||
tgggcatggaaactgctttg | |||
gatgatgacgacagccacac | |||
gggtacgtttgaagttactg | |||
gtgttgtattcgctgccaac | |||
ggtttcgcactgttcacgac | |||
tggctgctggaagaaacgcg | |||
ttcaatggtgacatcacgca | |||
catgcgcaacagcgttgaac | |||
ggcgttttcagtcagtatat | |||
atacgtttcaggtcgccttg | |||
tgagactccgccatcaactc | |||
gaaatcatcgcgcatagagg | |||
caatttgcggcacggtgatt | |||
ttgacgatttctaccagagt | |||
acctttgtcgccaatcacag | |||
ggatcagcgcgacgatacag | |||
atattgttcagcgacagctt | |||
gtctctttaatacctgtttt | |||
ctccttgtgatggtttacaa |