Name: probing mrna kinetics in space and time in escherichia coli using two-color single-molecule fluorescence in situ hybridization
Uploaded: 2020-07-30T13:30:00
Duration: 10 min 1 s
Description: Watch this Scientific Journal Video about Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization at JoVE.com

This protocol was developed by S.K. during her postdoctoral research in Dr. Christine Jacobs-Wagner&#39;s laboratory at the Howard Hughes Medical Institute and the Microbial Sciences Institute at Yale University. We thank Dr. Jacobs-Wagner and her lab members for various inputs during the method development and Laura Troyer for critical reading of the manuscript. S.K. acknowledges support from the Searle Scholars Program; K.V. acknowledges the support of James Scholar Preble Research Award from the University of Illinois.

<ol>
	<li>Bervoets, I., Charlier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity,+versatility+and+complexity+of+bacterial+gene+regulation+mechanisms:+opportunities+and+drawbacks+for+applications+in+synthetic+biology.">Diversity, versatility and complexity of bacterial gene regulation mechanisms: opportunities and drawbacks for applications in synthetic biology.</a> FEMS Microbiology Reviews. 43 (3), 304-339 (2019).</li><li>Epshtein, V., Nudler, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperation+between+RNA+polymerase+molecules+in+transcription+elongation.">Cooperation between RNA polymerase molecules in transcription elongation.</a> Science. 300 (5620), 801-805 (2003).</li><li>Vogel, U., Jensen, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA+chain+elongation+rate+in+Escherichia+coli+depends+on+the+growth+rate.">The RNA chain elongation rate in Escherichia coli depends on the growth rate.</a> Journal of Bacteriology. 176 (10), 2807-2813 (1994).</li><li>Tennyson, C. N., Klamut, H. J., Worton, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+dystrophin+gene+requires+16+hours+to+be+transcribed+and+is+cotranscriptionally+spliced.">The human dystrophin gene requires 16 hours to be transcribed and is cotranscriptionally spliced.</a> Nature Genetics. 9, 184 (1995).</li><li>Singh, J., Padgett, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rates+of+in+situ+transcription+and+splicing+in+large+human+genes.">Rates of in situ transcription and splicing in large human genes.</a> Nature Structural &amp; Molecular Biology. 16, 1128 (2009).</li><li>Selinger, D. W., Saxena, R. M., Cheung, K. J., Church, G. M., Rosenow, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+RNA+Half-Life+Analysis+in+Escherichia+coli+Reveals+Positional+Patterns+of+Transcript+Degradation.">Global RNA Half-Life Analysis in Escherichia coli Reveals Positional Patterns of Transcript Degradation.</a> Genome Research. 13 (2), 216-223 (2003).</li><li>Bernstein, J. A., Khodursky, A. B., Lin, P. -. H., Lin-Chao, S., Cohen, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+mRNA+decay+and+abundance+in+Escherichia+coli+at+single-gene+resolution+using+two-color+fluorescent+DNA+microarrays.">Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays.</a> Proceedings of the National Academy of Sciences. 99 (15), 9697-9702 (2002).</li><li>P&#233;rez-Ort&#237;n, J. E., Medina, D. A., Ch&#225;vez, S., Moreno, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+do+you+mean+by+transcription+rate.">What do you mean by transcription rate.</a> BioEssays. 35 (12), 1056-1062 (2013).</li><li>Tang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA-Seq+whole-transcriptome+analysis+of+a+single+cell.">mRNA-Seq whole-transcriptome analysis of a single cell.</a> Nature Methods. 6 (5), 377-382 (2009).</li><li>Kuchina, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+single-cell+RNA+sequencing+by+split-pool+barcoding.">Microbial single-cell RNA sequencing by split-pool barcoding.</a> BioRxiv. , 869248 (2019).</li><li>Blattman, S. B., Jiang, W., Oikonomou, P., Tavazoie, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prokaryotic+single-cell+RNA+sequencing+by+in+situ+combinatorial+indexing.">Prokaryotic single-cell RNA sequencing by in situ combinatorial indexing.</a> Nature Microbiology. , (2020).</li><li>Femino, A., Fay, F., Fogarty, K., Singer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+single+RNA+transcripts+in+situ.">Visualization of single RNA transcripts in situ.</a> Science. 280 (5363), 585-590 (1998).</li><li>Raj, A., vanden Bogaard, P., Rifkin, S. A., van Oudenaarden, A., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+individual+mRNA+molecules+using+multiple+singly+labeled+probes.">Imaging individual mRNA molecules using multiple singly labeled probes.</a> Nature Methods. 5 (10), 877-879 (2008).</li><li>Montero Llopis, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+organization+of+the+flow+of+genetic+information+in+bacteria.">Spatial organization of the flow of genetic information in bacteria.</a> Nature. 466 (7302), 77-81 (2010).</li><li>So, L. -. h., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+properties+of+transcriptional+time+series+in+Escherichia+coli.">General properties of transcriptional time series in Escherichia coli.</a> Nature Genetics. 43 (6), 554-560 (2011).</li><li>Taniguchi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+E.+coli+proteome+and+transcriptome+with+single-molecule+sensitivity+in+single+cells.">Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells.</a> Science. 329 (5991), 533-538 (2010).</li><li>Jones, D. L., Brewster, R. C., Phillips, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promoter+architecture+dictates+cell-to-cell+variability+in+gene+expression.">Promoter architecture dictates cell-to-cell variability in gene expression.</a> Science. 346 (6216), 1533-1536 (2014).</li><li>Iyer, S., Park, B. R., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absolute+quantitative+measurement+of+transcriptional+kinetic+parameters+in+vivo.">Absolute quantitative measurement of transcriptional kinetic parameters in vivo.</a> Nucleic Acids Research. 44 (18), 142 (2016).</li><li>Kim, S., Beltran, B., Irnov, I., Jacobs-Wagner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-Distance+Cooperative+and+Antagonistic+RNA+Polymerase+Dynamics+via+DNA+Supercoiling.">Long-Distance Cooperative and Antagonistic RNA Polymerase Dynamics via DNA Supercoiling.</a> Cell. 179 (1), 106-119 (2019).</li><li>Wang, M., Zhang, J., Xu, H., Golding, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+transcription+at+a+single+gene+copy+reveals+hidden+drivers+of+bacterial+individuality.">Measuring transcription at a single gene copy reveals hidden drivers of bacterial individuality.</a> Nature Microbiology. 4 (12), 2118-2127 (2019).</li><li>Joo, C., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Labeling DNA (or RNA) for single-molecule FRET. 2012 (9), 1005-1008 (2012).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Standard Ethanol Precipitation of DNA in Microcentrifuge Tubes. 2006 (1), 4456 (2006).</li><li>Skinner, S. O., Sep&#250;lveda, L. A., Xu, H., Golding, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+mRNA+copy+number+in+individual+Escherichia+coli+cells+using+single-molecule+fluorescent+in+situ+hybridization.">Measuring mRNA copy number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization.</a> Nature Protocols. 8 (6), 1100-1113 (2013).</li><li>Adesnik, M., Levinthal, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+synthesis+and+degradation+of+lactose+operon+messenger+RNA+in+E.+coli.">The synthesis and degradation of lactose operon messenger RNA in E. coli.</a> Cold Spring Harbor Symposia on Quantitative Biology. 35, 451-459 (1970).</li><li>Campbell, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+mechanism+for+rifampicin+inhibition+of+bacterial+RNA+polymerase.">Structural mechanism for rifampicin inhibition of bacterial RNA polymerase.</a> Cell. 104 (6), 901-912 (2001).</li><li>Raj, A., Tyagi, S., Walter, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Enzymology. 472, 365-386 (2010).</li><li>Sliusarenko, O., Heinritz, J., Emonet, T., Jacobs-Wagner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput,+subpixel+precision+analysis+of+bacterial+morphogenesis+and+intracellular+spatio-temporal+dynamics.">High-throughput, subpixel precision analysis of bacterial morphogenesis and intracellular spatio-temporal dynamics.</a> Molecular Microbiology. 80 (3), 612-627 (2011).</li><li>Paintdakhi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oufti:+an+integrated+software+package+for+high-accuracy,+high-throughput+quantitative+microscopy+analysis.">Oufti: an integrated software package for high-accuracy, high-throughput quantitative microscopy analysis.</a> Molecular Microbiology. 99 (4), 767-777 (2016).</li><li>Moffitt, J. R., Zhuang, X., Filonov, G. S., Jaffrey, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Enzymology. 572, 1-49 (2016).</li><li>Yu, J., Xiao, J., Ren, X., Lao, K., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+gene+expression+in+live+cells,+one+protein+molecule+at+a+time.">Probing gene expression in live cells, one protein molecule at a time.</a> Science. 311 (5767), 1600-1603 (2006).</li><li>Chen, H., Shiroguchi, K., Ge, H., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+study+of+mRNA+degradation+and+transcript+elongation+in+Escherichia+coli.">Genome-wide study of mRNA degradation and transcript elongation in Escherichia coli.</a> Molecular Systems Biology. 11 (1), 781 (2015).</li><li>Vogel, U., S&#248;rensen, M., Pedersen, S., Jensen, K. F., Kilstrup, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreasing+transcription+elongation+rate+in+Escherichia+Coli+exposed+to+amino+acid+starvation.">Decreasing transcription elongation rate in Escherichia Coli exposed to amino acid starvation.</a> Molecular Microbiology. 6 (15), 2191-2200 (1992).</li><li>Yang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+and+translation+contribute+to+gene+locus+relocation+to+the+nucleoid+periphery+in+E.+coli.">Transcription and translation contribute to gene locus relocation to the nucleoid periphery in E. coli.</a> Nature Communications. 10 (1), 5131 (2019).</li><li>Zenklusen, D., Larson, D. R., Singer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-RNA+counting+reveals+alternative+modes+of+gene+expression+in+yeast.">Single-RNA counting reveals alternative modes of gene expression in yeast.</a> Nature Structural &amp; Molecular Biology. 15 (12), 1263-1271 (2008).</li><li>Fontenete, S., Guimar&#227;es, N., Wengel, J., Azevedo, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+melting+temperatures+in+fluorescence+in+situ+hybridization+(FISH)+procedures+using+thermodynamic+models.">Prediction of melting temperatures in fluorescence in situ hybridization (FISH) procedures using thermodynamic models.</a> Critical Reviews in Biotechnology. 36 (3), 566-577 (2016).</li><li>Sep&#250;lveda, L. A., Xu, H., Zhang, J., Wang, M., Golding, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+gene+regulation+in+individual+cells+reveals+rapid+switching+between+promoter+states.">Measurement of gene regulation in individual cells reveals rapid switching between promoter states.</a> Science. 351 (6278), 1218-1222 (2016).</li><li>Huang, B., Wang, W., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Super-Resolution+Imaging+by+Stochastic+Optical+Reconstruction+Microscopy.">Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy.</a> Science. 319 (5864), 810-813 (2008).</li><li>Moffitt, J. R., Pandey, S., Boettiger, A. N., Wang, S., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+organization+shapes+the+turnover+of+a+bacterial+transcriptome.">Spatial organization shapes the turnover of a bacterial transcriptome.</a> eLife. 5, 13065 (2016).</li><li>Fei, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+in+vivo+target+search+kinetics+of+regulatory+noncoding+RNA.">Determination of in vivo target search kinetics of regulatory noncoding RNA.</a> Science. 347 (6228), 1371-1374 (2015).</li></ol>

The authors declare they have no competing financial interests.

Here, we presented a smFISH protocol for measuring mRNA kinetics in E. coli. In the previously published smFISH protocols for bacteria23, cells were kept in the tubes until the very end of the protocol, that is until they are ready for imaging. While it has many benefits, such as minimal nonspecific binding of fluorescent probes on the coverslip surface23, it is difficult to follow these protocols when there are&#160;many samples&#160;from a time-course experiment. First, a relatively large volume of cells (&#62;1 mL) needs to be sampled and even harvested before fixation. Second, the cell samples need to be centrifuged multiple times to exchange solutions and to wash after the hybridization step. In our protocol, a small volume (&#60;1 mL) of culture is directly mixed with a fixing solution in a 1.5-mL tube, helping to quickly &#8220;freeze&#8221; the cell state at the moment of sampling. Also, cells stay attached to the surface throughout the procedure, and different solutions can be exchanged quickly by aspirating liquids with a vacuum filtration system and applying solution drops at once with a multi-channel pipette. This difference makes our protocol highly advantageous when a large number of samples need to be processed at once. Using our protocol, 12-48 samples can be handled simultaneously and the entire FISH procedure can be completed within ~8 hours, about a similar amount of time needed for a few samples (Figure 2). Although we used the expression of lacZ in E. coli as an example, the protocol is widely applicable to different genes and bacterial species with considerations discussed below.
For different genes, the first thing to consider is smFISH probes. One may design oligonucleotide probes that tile the mRNA of interest (Figure 1A)13. In this &#8220;tiling&#8221; probe approach, each probe is ~20 base long and labeled with a fluorophore at the 5&#8217; or 3&#8217; terminus. This strategy is convenient as no genetic manipulation is needed. Alternatively, a tandem repeat&#160;of ~20 bp sequence, foreign to the genomic sequence (e.g., an array of lacO sequence in Caulobacter crescentus14), may be inserted in the untranslated region of a gene of interest and a single probe complementary to the repeat unit is used to label the mRNA (&#8220;array&#8221; approach; Figure 1B). In both cases, multiple fluorophores decorate an mRNA, giving amplified fluorescence signal that can be easily differentiated from a single probe nonspecifically bound inside a cell.
Whether to choose &#8220;tiling&#8221; or &#8220;array&#8221; approaches depends on the negative control, a sample where nonspecific binding of probes is tested because it lacks the target mRNA. For tiling probes (Figure 1A), a mutant strain without the gene of interest or a condition, in which the gene is not transcribed (e.g., repression of lacZ) can serve as a negative control for testing nonspecific binding of probes. For the array-based smFISH (Figure 1B), a wild-type strain lacking the array can serve as a negative control because it does not contain binding sites for the probes.
Optimal hybridization conditions may depend on the probe sequences and even the choice of fluorophore dyes. We optimized the hybridization condition for lacZ probe sets by keeping the hybridization temperature at 37 &#176;C and testing different concentrations of probe sets and formamide in the hybridization solution. Higher concentrations of formamide tend to reduce both nonspecific and specific binding26,35. We recommend systematically changing the hybridization and its wash conditions while keeping hybridization time and temperature the same. As the condition becomes more stringent, both nonspecific and specific binding decrease (Figure 6). It is important to find a point where the nonspecific binding starts to hit below an acceptable threshold without further compromising specific binding. For example, we used the signal level obtained without any probes (&#8220;no probes&#8221;) as a threshold (Figure 6).
The two-color smFISH method labeling two separate regions of an mRNA is limited to long genes. To&#160;measure&#160;the rate of transcription elongation, we took advantage of the fact that lacZ is long (3075 bp) and its expression can be induced by IPTG. When a gene is short, it is difficult to design two tiling probe sets (near 5&#8217; and 3&#8217; ends) and resolve the time delay between appearances of 5&#8217; vs. 3&#8217; mRNA regions. In this case, one may count nascent mRNAs at steady state by smFISH and analyze their distribution with an analytical model that has the rate of transcription elongation as a fitting parameter20. Also, when a gene of interest is not inducible, one may treat cells with rifampicin at time zero and measure the temporal change in 5&#8217; and 3&#8217; mRNA sub-regions. The delay from the decrease of 5&#8217; mRNA signal to that of 3&#8217; mRNA signal can then be used to calculate the rate of transcription elongation as done previously31.
Finally, the smFISH protocol is versatile and can be combined with other labeling schemes. Previously, DNA locus was visualized together with mRNAs by combining mRNA FISH with either DNA FISH14 or fluorescent reporter-operator system20. Protein products may&#160;be visualized by performing immunofluorescence together with mRNA FISH14,36. Also, it can be combined with three-dimensional super-resolution microscopy37 to visualize mRNAs in all three dimensions38,39.&#160;

The flow of genetic information from DNA to mRNA and protein is one of the most fundamental cellular processes, whose regulation is important for cellular fitness1. The number of mRNAs in a cell is determined by two dynamic processes, transcription, and mRNA degradation. However, how transcription and mRNA degradation are regulated in time and space of a single cell remains not completely understood, largely due to the shortage of experimental methods to quantitatively measure their kinetics in vivo.
Methods based on total mRNAs extracted from a population of cells, such as Northern blot, RT-PCR, RNA sequencing, and gene expression microarrays, can measure the relative difference in mRNA levels and have been widely used to analyze the rate of transcription elongation2,3,4,5 or the rate of mRNA degradation6,7. However, they do not provide the absolute number of mRNAs per cell, and hence, they are not suitable for probing the rate of transcription initiation8. Also, because mRNAs are extracted from a population of cells, the spatial distribution of mRNAs within a single cell and the variability of mRNA copy numbers among cells cannot be measured.
Next-generation RNA sequencing on individual cells (scRNAseq) can quantify the number of mRNAs per cell in a genomic scale9. However, it remains difficult to use this technique to measure transcription kinetics, due to challenges with sample preparation and high cost. In particular, the application of scRNAseq to bacteria has been technically difficult due to low mRNA abundance10,11.
Single-molecule fluorescence in situ hybridization (smFISH) is based on the hybridization of fluorescently-labeled single-stranded probes whose sequences are complementary to the target mRNA of interest12,13. The concept of sequence-specific hybridization is similar to that used in Northern blot or RT-PCR, but the hybridization is done in situ within fixed cells, to preserve the native localization of mRNAs. The signal of a single mRNA is amplified using many probes, ~20 nucleotides (nt) in length, hybridizing to different parts of an mRNA (Figure 1A)13. In this &#8220;tiling&#8221; probe approach, the number of probes needed to detect a single mRNA sets a lower limit on the length of mRNA that can be assayed. Alternatively, the mRNA of interest may be transcriptionally fused to a non-coding array of tandem Lac operator sequences, such that multiple copies of a fluorescently labeled lacO probe hybridize to&#160;a single mRNA (Figure 1B)14.
smFISH has been used to quantify the number of mRNAs per cell at steady state (i.e., when synthesis and decay are in balance) and to analyze the mean and variability of mRNAs among bacterial cells15,16,17. Recently, smFISH has been applied to quantify mRNA numbers at non-steady state, right after induction or repression of gene expression in E. coli18,19,20. The temporal changes in the absolute mRNA copy numbers were then used to calculate the rate of transcription initiation, elongation, and termination, as well as the rate of mRNA degradation. For this application, conventional smFISH procedures can be cumbersome because there are many samples, each representing one time point, that need to go through multiple buffer exchange steps (i.e., centrifugation and washing). Here, we describe an smFISH protocol, in which the sample handling steps are dramatically simplified by having cells adhered to the surface of a coverslip and by aspirating liquids with a vacuum filtration system14,19. Using the expression of lacZ in E. coli as an example, the full workflow (Figure 2) is demonstrated, including image analysis (Figure 3) yielding the in vivo kinetics of transcription (initiation, elongation, and termination) and mRNA degradation, cell-to-cell variability in mRNA expression, and mRNA localization. We anticipate that the protocol is widely applicable to probe in vivo kinetics and localization of other mRNAs in various bacteria species.

probing mrna kinetics in space and time in escherichia coli using two-color single-molecule fluorescence in situ hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Watch this Scientific Journal Video about Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization at JoVE.com

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...

Research

JoVE Journal

Biology

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Fluorescence in situ hybridization FISH Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

Measuring the Kinetics of mRNA Transcription in Single Living Cells

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...