This research was supported by grants GM046406 (to D.B.) and by the National Institute of General Medical Sciences Center for Quantitative Biology (GM071508). R.S.M. acknowledges funding from the NSF Graduate Research Fellowship. M.N.M. is supported by a Lewis-Sigler Fellowship. We would like to acknowledge members of the Botstein lab for helpful discussions and the former members Allegra Petti and Nikolai Slavov for their contributions to the metabolic cycle project. We thank Daniel Zenklusen and Robert Singer for getting us started with the FISH method.

Figures 1 and 2 are schematics of the FISH experimental procedures and image analysis pipeline used for quantifying FISH images.
1. Solutions to Prepare
*The solutions below are for use with Singer probes. If using Stellaris probes, replace "40% formamide" with "10% formamide" in both Hybridization Buffer and Wash Buffer. Additional changes to Hybridization Buffer when using Stellaris probes are (1) add 1 g Dextran Sulfate and (2) do not include 10 mg ssDNA.
Buffer B
8 ml 1 M KH2PO4 
41.5 ml 1M K2HPO4 
109.3 g Sorbitol
Spheroplasting Buffer
890 &mu;l Buffer B 
100 &mu;l VRC 
10 &mu;l 25,000 U/ml Lyticase 
2 &mu;l &beta;-mercaptoethanol
Hybridization Buffer (10 ml, final volume)
10 mg E. coli tRNA 
10 mg ssDNA* 
100 &mu;l 200 mM VRC stock 
40 &mu;l of 50 mg/ml BSA 
1 ml 20X SSC 
4 ml 40% Formamide* 
Nuclease Free Water (to 10 ml final volume) 
1 g Dextran Sulfate*
*Hybridization Buffer may be kept in 0.5 ml aliquots at -20 &deg;C for convenience.
Wash Buffer (50 ml, final volume)
5 ml 20X SSC 
20 ml 40% Formamide* 
Nuclease Free Water (to 50 ml final volume)
Labeling Buffer
1.06 g Sodium Carbonate 
100 ml DEPC Water 
pH 9
 
2. Probe-labeling (Singer Probes Only)
We obtain these probes by in-house synthesis using an ABI oligonucleotide synthesis apparatus. Typically, 4-5 ~50 bp oligonucleotides are synthesized that are homologous to the gene of interest, substituting amino-allyl thymidine for several thymidines spaced at least 8, preferably 10+ bp apart. Because of their sensitivity to ozone, we work in an ozone-free facility when using CY dyes.
<ol>
	<li>Obtain ~5 probes and resuspend in 100 &mu;l water - check concentrations on Nanodrop.</li>
	<li>Depending on how many probes/gene, combine total of 10 &mu;g oligonucleotides/gene (e.g. if have 5 probes/gene, then want 2 &mu;g/probe).</li>
	<li>Use QIAquick columns to purify probes according to QIAquick Nucleotide Removal Kit Protocol.</li>
	<li>Add 10 volumes Buffer PN to total volume of combined probes and mix.</li>
	<li>Apply sample to QIAquick column - if total volume is greater than 750 &mu;l, spin down twice using half of volume in each spin</li>
	<li>Let stand for 1 min.</li>
	<li>Centrifuge 1 min at 6,000 rpm.</li>
	<li>Wash with 750 &mu;l Buffer PE.</li>
	<li>Centrifuge 1 min at 6,000 rpm.</li>
	<li>Dispose of flow through and re-spin column for 1 min at 13,000 rpm to dry.</li>
	<li>Place QIAquick column in new microcentrifuge tube and elute DNA with 50 &mu;l H2O - Make sure H2O pH is within 7.0 and 8.5 and is placed directly on membrane.</li>
	<li>Let stand for 1 min.</li>
	<li>Centrifuge for 1 min at 13,000 rpm to elute DNA.</li>
	<li>Lyophilize DNA at 45 &deg;C.</li>
	<li>Resuspend pellet in 10 &mu;l labeling buffer and add to tube of dye without touching the dye.</li>
	<li>Repeat with another 10 &mu;l labeling buffer.</li>
	<li>Vortex and spin down tube of dye and DNA.</li>
	<li>Cover tubes with aluminum foil and keep in dark at RT O/N to label.</li>
	<li>Repeat QIAquick Nucleotide Removal Kit Protocol. Perform with two differences: 
	- Add 200 &mu;l PN buffer to probes and put labeled probes through columns 2x. 
	- Perform 3 washes with buffer PE in order to wash away any unattached dye before elution.</li>
	<li>After elution obtain concentration via nanodrop. The labeling efficiency is typically ~0.25 pmol/ng of single-stranded DNA.</li>
</ol>
3. Coverslip Preparation
<ol>
	<li>Place coverslips on slides in Plasma-preen vacuum chamber (<a href="http://www.plasmapreen.com/" target="_blank">http://www.plasmapreen.com/</a>) (the closer to the center the better).</li>
	<li>Put vacuum chamber in microwave and make sure it is sealed.</li>
	<li>Turn on pump first then turn on vacuum only once pump is started.</li>
	<li>Turn on microwave, and stop 5 sec after plasma is visible.</li>
	<li>Turn off vacuum then pump.</li>
	<li>Pull out vacuum chamber and remove coverslips with forceps (those that have fallen need to be cleaned again).</li>
	<li>Place coverslips cleaned side up in 12-well plates.</li>
</ol>
4. Fixation Procedure
<ol>
	<li>Grow yeast to an OD600 of around 0.1-0.2 in minimal media. 10 ml of cells yields enough for ~10 separate hybridizations.</li>
	<li>Add 1/10 volume 37% formaldehyde directly to the growth media (10 ml of culture + 1 ml 37% formaldehyde) and let sit for 45 min.</li>
	<li>Wash 2x with 1 ml ice cold Buffer B in a microcentrifuge tube (can spin at 13,000 rpm for 1 min).</li>
	<li>Add 1 ml of spheroplasting buffer.</li>
	<li>Incubate at 37 &deg;C for 15 min. Check cells every few minutes under microscope until most cells are black (i.e. not phase-bright).</li>
	<li>Wash 2x with ice cold Buffer B, spinning at low speed (~3,500 rpm).</li>
	<li>Add 1 ml of 70% EtOH, resuspend gently and leave overnight at 4 &deg;C (can store indefinitely at -20 &deg;C).</li>
</ol>
5. Hybridization Procedure
<ol>
	<li>Prepare the hybridization solution: to 100 &mu;l of hybridization buffer, add 1-3 &mu;l of probe, then vortex and centrifuge. Singer probes use 8-10 ng total per probe set (i.e. per gene). We have imaged up to 3 genes simultaneously with 3 different labeled probe sets.</li>
</ol>
Be sure to warm the hybridization solution to room temperature before opening it.
For Stellaris probes, it is recommended to start 4 separate hybridization reactions by adding 1 &mu;l each of 1:10, 1:20, 1:50 and 1:100 working dilutions of probes to see which one is optimal. Working dilutions of Stellaris probes are prepared in Hybridization buffer.
<ol start="2">
	<li>Centrifuge (for all subsequent steps; 3,500 rpm; 5 min) the fixed cells (e.g. 200 &mu;l) and aspirate away the ethanol.</li>
	<li>Gently resuspend in 1 ml wash buffer that contains the same percentage formamide as the hybridization buffer. Let stand for 2-5 min.</li>
	<li>Centrifuge sample and aspirate wash buffer, then add hybridization solution. Incubate in the dark with gentle shaking, O/N at 37 &deg;C.</li>
</ol>
Note: the following procedure is for applying/imaging cells on coverslips. For washing/imaging cells in 96-well plates, including alternative reactive oxygen species scavenger solution see <a href="http://www.biosearchtech.com/stellarisprotocols" target="_blank">http://www.biosearchtech.com/stellarisprotocols.</a>
<ol start="5">
	<li>The next day, or in advance, clean (see procedure) and treat cover slips with 150 &mu;l 0.01% Poly-L-Lysine for 5 min. Aspirate, let dry, wash 3x with dH2O and let dry.</li>
	<li>In the morning, add 1 ml of wash buffer to the sample, gently resuspend, centrifuge and aspirate, then resuspend in another 1 ml of wash buffer and incubate at 37 &deg;C for 30 min.</li>
	<li>Wash with 2X SSC + 0.1% Triton X-100 at RT 15 min on shaker.</li>
	<li>Take mounting medium out of freezer to allow it to come up to RT before mounting.</li>
	<li>Wash with 1X SSC at RT 15 min on shaker.</li>
	<li>Dilute DAPI into PBS (0.1 &mu;g/ml final) and resuspend cells in 150 &mu;l.</li>
	<li>Place solution onto cleaned/poly-lys-treated cover slips in 12-well plate; at least 30 min undisturbed.</li>
	<li>Remove solution (you may place it on a spare cover slip as a backup) and wash 3x with 1 ml 1X PBS.</li>
	<li>Place 3 &mu;l of Prolong Gold mounting medium onto a slide (Invitrogen P36934). Do this one at a time if you have multiple cover slips to avoid drying of mounting medium.</li>
	<li>Add ~0.5 ml ethanol to coverslip in 12-well plate, remove the cover slip and air dry while holding with tweezers.</li>
	<li>Place cover slip cell-side down onto mounting medium and let harden several hours or O/N in the dark.</li>
	<li>Seal the edges with nail polish and proceed to imaging.</li>
</ol>
6. Imaging of Cells with Olympus IX-81 Inverted Microscope Overview
<ol>
	<li>For image acquisition we utilize Slidebook (intelligent-imaging.com) software and a 100X, 1.45 NA, TIRFM oil objective. For serial GFP, DAPI, Cy3, Cy3.5 and Cy5 imaging: Chroma filter sets (see reagents).</li>
	<li>Use DAPI filter to find and focus cells.</li>
	<li>Set this as the reference point.</li>
	<li>Take a z-stack of images around the reference point where the total distance is 5 &mu;M with 0.2 &mu;M size (25 planes). Repeat for each dye channel (i.e. each probe set).</li>
	<li>Export as a 16-bit TIFF for image analysis.</li>
</ol>
7. Image Analysis Overview (Singer Probes)
Below we provide an outline of computational methods we use for analyzing FISH images in MATLAB. The relevant MATLAB functions used are bracketed on the right. The algorithms and thresholds are currently tuned for data from Singer style probes. Using Stellaris style probes will require some adjustment, particularly to the final filtering step (7.8).
Cell Identification15
<ol>
	<li>Separate cells from background using a global threshold on DAPI images16 [graythresh].</li>
	<li>Identify nuclei using extended maxima function [imextendedmax].</li>
	<li>Segment cells using nuclei as seeds for a watershed algorithm [watershed].</li>
</ol>
Find spots in each fluorescence channel
<ol start="4">
	<li>Perform tophat transformation to normalize background and improve signal to noise ratio [bwmorph].</li>
	<li>Find maxima to identify the in-focus layer (in the z-plane) for each spot [imregionalmax].</li>
	<li>Filter potential spots using a linear fit model to a radial gradient.</li>
</ol>
Measure spot intensity and filter single vs. multiple probe signals
<ol start="7">
	<li>Fit a 2D Gaussian profile to the spot in the previously identified in-focus layer and estimate intensity17.</li>
	<li>Filter out weak spots using a threshold (based on histogram). For Singer type probes this is important, but is less so for Stellaris probes.</li>
	<li>Count spots in each cell.</li>
</ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+and+Nuclear+Location+of+Mouse+Satellite+DNA+in+Individual+Cells.">Chromosomal and Nuclear Location of Mouse Satellite DNA in Individual Cells.</a> Nature. 225, 912 (1970).</li><li>Bauman, J. G., Wiegant, J., Borst, P., van Duijn, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+fluorescence+microscopical+localization+of+specific+DNA+sequences+by+in+situ+hybridization+of+fluorochromelabelled+RNA.">A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochromelabelled RNA.</a> Exp Cell Res. 128, 485-490 (1980).</li><li>Singer, R. H., Ward, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+gene+expression+visualized+in+chicken+muscle+tissue+culture+by+using+in+situ+hybridization+with+a+biotinated+nucleotide+analog.">Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with a biotinated nucleotide analog.</a> Proc. Natl. Acad. Sci. U.S.A. 79, 7331-7335 (1982).</li><li>Silverman, S. 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=Metabolic+cycling+in+single+yeast+cells+from+unsynchronized+steady-state+populations+limited+on+glucose+or+phosphate.">Metabolic cycling in single yeast cells from unsynchronized steady-state populations limited on glucose or phosphate.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 6946-6951 (2010).</li><li>Trcek, T., Larson, D. R., Moldon, A., Query, C. 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The authors declare they have no competing financial interests.

To date, FISH has primarily been a low-throughput method. The use of Cy3, Cy3.5, and Cy5 dyes limits the number of genes one can investigate in single cells to three at a time. Some additional probes have been developed (Stellaris) but the number of distinguishable probes is still at most seven. To circumvent this limitation, combinatorial labeling strategies using multiple fluorophores have been used to create barcodes for different mRNA species19,20. Most recently, Lubeck and Cai used optical and spectral barcoding to quantify 32 different species simultaneously with FISH in single yeast cells19. One limitation of this recent combinatorial approach is it requires the use of super-resolution microscopy. The analysis needed to distinguish the barcoded probes is also quite complex.
We have found that Cy3 and Cy3.5 are preferable to Cy5 for FISH experiments. One of the limitations of the Cy5 dye is its sensitivity to photobleaching. However, Stellaris has recently developed Cy5 variants that are advertised as more resistant to photobleaching, and may alleviate this technical issue. It also worth noting that FISH is an expensive method to implement and that both Singer and Stellaris probes typically cost $700 - $1,000 per probe set, although prices for commercially available probes should decrease in the future. Sparing of reagents and efficient labeling brings Singer probes down to the lower range in price.
One of the major technical challenges is the separation of single versus multiple probe spots, which requires the implementation of sophisticated spot-determining algorithms. This can take extensive manual review to tune image analysis parameters for specific experimental setups. An outline of our computational pipeline with relevant MATLAB functions is provided in Section 7 of the protocol. This issue is somewhat alleviated by the Stellaris probes which have only one label per probe. It therefore requires the colocalization of multiple probes to see a signal.
Because FISH necessitates fixing cells, it does not facilitate tracking individual cells over time. Previously, we used FISH snapshot data to reconstruct the dynamics of gene expression in individual metabolically cycling yeast populations6. Metabolic cycling is observed in pre-starved, continuous cultures, and is characterized by population-wide collective oscillations in oxygen consumption. These oscillations are associated with genome-wide oscillations of transcripts that occur for half of all yeast genes at different phases of oxygen consumption. We sought to determine if metabolic cycling was present in unsynchronized continuous yeast cultures. If present, transcripts that are anti-correlated in synchronous populations should also be anti-correlated in unsynchronized single cells, and vice versa for correlated transcripts.
To reconstruct dynamics of mRNA production in time, the observed snapshot data must be compared to what is expected from a model of the underlying behavior. There are theoretical limitations to when such "snapshots" of gene expression data can be used to determine the underlying gene expression dynamics and which kinds of models can be distinguished21. For the metabolic cycle data, rather than directly showing the presence of temporal oscillations, statistical measurements were implemented to substantiate that there is indeed a cell autonomous oscillatory program consistent with bulk microarray measurements.

Using bulk measurement techniques, it is not possible to assay the number of transcripts or transcriptional activity within single cells1. Using fluorescent proteins driven by promoters of interest as reporters of gene expression can address this issue to some extent, but the time required for fluorescent proteins to fold obscures early dynamics. Long-lived fluorescent proteins also cannot report mRNA lifetimes. The FISH method can be used to assay mRNA during its complete life cycle, from transcription initiation in the nucleus to subsequent maturation and decay in single cells, with single-molecule resolution.
The original in situ experiments for visualizing nucleic acids used radiolabeled RNA probes to probe DNA elements. These included visualizing ribosomal DNA in ovaries of the frog Xenopus laevis 2 and satellite DNA in mouse tissue3. The first fluorescent in situ experiment used an RNA molecule marked with a fluorophore to probe particular DNA sequences4. The first application of fluorescent probes for visualizing RNA in situ was the visualization of actin gene expression in chicken muscle tissue culture5. More recently, in budding yeast, FISH has been used to investigate oscillations in transcription during the yeast metabolic cycle6, the decay of mRNAs during cell cycle progression7, and spatial localization of mRNA transcripts during mitosis8. FISH has been used in yeast to show that uncorrelated fluctuations in constitutively transcribed genes, which constitute more than half of all yeast genes, arise from uncorrelated transcription initiation9. In non-yeast species, FISH has been used to identify stem-cell markers in the mouse intestine10 and to determine that incomplete penetrance of cell fates can result from stochastic gene expression fluctuations in C. elegans embryos11.
The FISH method described here works by hybridizing dye-labeled, single-stranded DNA probes to mRNA messages. Cells are imaged and mRNAs are counted using a spot-detecting algorithm. Single-stranded probes can be generated with a DNA synthesizer and then labeled (referred to here as Singer probes) or ordered commercially as pre-labeled probes (Stellaris probes)12,13. A major difference between the Singer and Stellaris probes is that the Singer probes are longer (~50 bp) and are multi-labeled while the Stellaris probes are short (~20 bp) with only one label per probe, as described by Raj et al 14. Additionally, the Stellaris approach uses many more probes per gene than that of Singer (~30 versus 5 probes per gene, respectively). Below we provide a protocol that describes the use of either type of probe. In section 2, we provide a protocol for labeling amino-allyl thymidine-containing probes with a chosen Cy dye. An overview of the computational steps required to identify single mRNA spots is provided in Section 7.

<table><tbody><tr><td>Vanadyl Ribonucleoside Complex</td><td>NEB</td><td>S1402S</td><td></td></tr><tr><td>Lyticase</td><td>Sigma</td><td>L5263</td><td></td></tr><tr><td>&lt;em&gt;E. coli &lt;/em&gt;tRNA</td><td>Roche</td><td>1010954001</td><td></td></tr><tr><td>BSA (RNase free)</td><td>Ambion</td><td></td><td></td></tr><tr><td>Beta-mercapt&amp;#8211;thanol</td><td>Fisher</td><td>03446l</td><td></td></tr><tr><td>DAPI, dilactate</td><td>Sigma</td><td>D9564</td><td></td></tr><tr><td>PBS 10X (RNase free)</td><td>Ambion</td><td>AM9624</td><td></td></tr><tr><td>Triton X-100</td><td>Shelton Scientific</td><td></td><td></td></tr><tr><td>Dextran sulfate</td><td>Sigma</td><td>D6001</td><td>Or equivalent</td></tr><tr><td>Saline-sodium citrate (SSC) 20X</td><td>VWR</td><td>82021-484</td><td></td></tr><tr><td>Formamide (deionized)</td><td>Ambion</td><td>AM9342</td><td></td></tr><tr><td>Nuclease-free water</td><td>Ambion</td><td>AM9932</td><td></td></tr><tr><td>Alpha-D-glucose</td><td>Sigma</td><td>158968</td><td>For GLOX solution</td></tr><tr><td>1 M Tris-HCl, pH 8.0</td><td>Ambion</td><td>AM9855G</td><td></td></tr><tr><td>100% Ethanol</td><td></td><td></td><td></td></tr><tr><td>Glucose oxidase</td><td>Sigma</td><td>G0543</td><td>For GLOX solution</td></tr><tr><td>Catalase</td><td>Sigma</td><td>C3155</td><td>For GLOX solution</td></tr><tr><td>Concanavalin A</td><td>MP Biomedicals</td><td>150710</td><td></td></tr><tr><td>Polylysine (0.01%)</td><td>Sigma</td><td>P8920</td><td></td></tr><tr><td>Coverslips</td><td>Warner Instruments</td><td>Cs-18R15</td><td></td></tr><tr><td>Prolong Gold Mounting Medium</td><td>Invitrogen</td><td>P36934</td><td></td></tr><tr><td>QIAquick Nucleotide Removal Kit</td><td>QIAGEN</td><td>28304</td><td></td></tr><tr><td>FISH Probes</td><td>Biosearch Technologies</td><td></td><td>Custom order for your desired mRNA sequence</td></tr><tr><td>Glass bottom 96-well plates</td><td>Nunc</td><td>265300</td><td>Alternative to coverslips</td></tr><tr><td>12-well plates</td><td>BD Falcon</td><td>351143</td><td></td></tr><tr><td>Cy3, Cy3.5, Cy5 dyes</td><td>GE Healthcare</td><td></td><td>monofunctional NHS-ester</td></tr><tr><td colspan="4">&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td></tr><tr><td>Plasma-Preen I Cleaner</td><td>Terra Universal</td><td>9505-00</td><td>Controller (Cat #9505-17 optional)</td></tr><tr><td>Vacuum Pump</td><td>Alcatel</td><td>205SDMLAM</td><td>For operating Plasma-Preen</td></tr><tr><td>Widefield Fluorescence Microscope</td><td>Olympus</td><td>IX81</td><td>Or equivalent</td></tr><tr><td>100X objective</td><td>Olympus</td><td>1-UB617R</td><td></td></tr><tr><td>Light Source</td><td>X-Cite</td><td>XCT 10-A</td><td>Or equivalent</td></tr><tr><td>Filter Sets</td><td>Chroma</td><td>&lt;em&gt;U-NSP100V2-SPR, U-NSP101V2-SPR, U-NSP102V2-SPR, U-NSP103V2-SPR,U-NSP104V2-SPR.&lt;/em&gt;</td><td></td></tr><tr><td>Cooled CCD or EMCCD Camera</td><td>Hamamatsu</td><td>C4742-98-24ER</td><td></td></tr></tbody></table>

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 using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Figure 3 shows typical histograms computed from FISH images and used to determine the number of mRNAs present in single cells. An important advantage of microscopy-based RNA quantification is that one can obtain information on the localization of transcripts. For example, we used FISH to identify mRNAs in single cells with an inducible CBF1 allele (Figure 4). Because many mRNA molecules are present at the site of transcription, we are able to identify the presence and location of transcription sites within the nucleus.
By utilizing different dyes to label mRNAs of different genes, one can quantify multiple mRNA species in the same cells. To demonstrate this, yeast cells were incubated in the presence of &alpha;-factor and sorbitol. FUS1 transcription (Quasar670 dye, red) is induced by &alpha;-factor. STL1 transcription (Quasar 570 dye, green) is induced by increases in extracellular osmolarity (Figure 5). Figure 4 is an example of FISH with the Singer probes. Figure 5 is an example of FISH with the Stellaris probes.
<img alt="Figure 1" src="/files/ftp_upload/50382/50382fig1.jpg" /> 
Figure 1. Schematic of FISH experimental procedure. <a href="https://www.jove.com/files/ftp_upload/50382/50382fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/50382/50382fig2.jpg" /> 
Figure 2. Schematic of image analysis pipeline. The final spots are determined in the rightmost figure.
<img alt="Figure 3" src="/files/ftp_upload/50382/50382fig3.jpg" /> 
Figure 3. Histogram of spot intensities for a particular gene using Singer probes. Probe intensities are computed both inside (red) and outside (blue) cells. Low intensity spots are either noise or single probes. Real mRNA messages are labeled with multiple probes. When using Stellaris probes, single probes are less detectable and thus the thresholds and filtering must be adjusted accordingly.
<img alt="Figure 4" src="/files/ftp_upload/50382/50382fig4.jpg" /> 
Figure 4. Representative results of Singer FISH procedure. In this experiment, CBF1 transcription is activated with an inducible promoter18. Nuclei are stained blue with DAPI. CBF1 mRNAs are tagged with Cy3-labeled probes. White arrows highlight the presence of CBF1 transcription sites in the nucleus. Single mRNA transcripts are visible in the cytoplasm.
<img alt="Figure 5" src="/files/ftp_upload/50382/50382fig5.jpg" /> 
Figure 5. Representative results of Stellaris FISH procedure. MATa yeast cells were simultaneously exposed to 30 ng/ml &alpha;-factor and 0.75 M sorbitol for 10 min and were simultaneously probed for FUS1 (Quasar 670, red) and STL1 (Quasar 570, green) transcripts. In the highlighted box, we can see one cell responding only to the pheromone (FUS1 start site, red) and another predominantly responding to sorbitol (STL1 start site, green).

Watch this Scientific Journal Video about Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae at JoVE.com

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

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

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 ...

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JoVE Journal

Biology

34.7K Views.  Princeton University. Dit protocol beschrijft een experimentele procedure voor het uitvoeren Fluorescentie In situ Hybridisatie (FISH) voor het tellen van mRNA's in enkele cellen in single-molecule resolutie.

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

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Genetics

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

Biochemistry

Visualizing Yeast Organelles with Fluorescent Protein Markers

The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have constructed fluorescent protein-based markers for major organelles and endomembrane structures, including the nucleus, endoplasmic reticulum (ER), Golgi apparatus, endosomes, vacuoles, mitochondria, peroxisomes, lipid droplets, and autophagosomes. The protocol presented here describes the procedures for using these markers in yeast, including DNA preparation for yeast transformation, selection and evaluation of transformants, fluorescent microscopic observation, and the expected outcomes. The text is geared toward researchers who are entering the field of yeast organelle study from other backgrounds. Essential steps are covered, as well as technical notes about microscope hardware considerations and several common pitfalls. It provides a starting point for people to observe yeast subcellular entities by live-cell fluorescent microscopy. These tools and methods can be used to identify protein subcellular localization and track organelles of interest in time-lapse imaging.

Here we describe the use of a set of fluorescent protein-based organelle markers in live-cell imaging of the budding yeast, Saccharomyces cerevisiae.

The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

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).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

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.

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

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