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>

<ol>
	<li>Femino, A. M., Fay, F. S., Fogarty, K., 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=Visualization+of+single+RNA+transcripts+in+situ.">Visualization of single RNA transcripts in situ.</a> Science. 280, 585-590 (1998).</li><li>Gall, J. G., Pardue, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+Detection+of+Rna-DNA+Hybrid+Molecules+in+Cytological+Preparations.">Formation and Detection of Rna-DNA Hybrid Molecules in Cytological Preparations.</a> Proceedings of the National Academy of Sciences of the United States of America. 63, 378 (1969).</li><li>Jones, K. 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. C., 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-molecule+mRNA+decay+measurements+reveal+promoter-+regulated+mRNA+stability+in+yeast.">Single-molecule mRNA decay measurements reveal promoter- regulated mRNA stability in yeast.</a> Cell. 147, 1484-1497 (2011).</li><li>Bertrand, E., 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=Localization+of+ASH1+mRNA+particles+in+living+yeast.">Localization of ASH1 mRNA particles in living yeast.</a> Mol Cell. 2, 437-445 (1998).</li><li>Gandhi, S. J., Zenklusen, D., Lionnet, T., 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=Transcription+of+functionally+related+constitutive+genes+is+not+coordinated.">Transcription of functionally related constitutive genes is not coordinated.</a> Nat. Struct. Mol. Biol. 18, 27-34 (2011).</li><li>Itzkovitz, 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=Single-molecule+transcript+counting+of+stem-cell+markers+in+the+mouse+intestine.">Single-molecule transcript counting of stem-cell markers in the mouse intestine.</a> Nature Cell Biology. 14, 106-U193 (2012).</li><li>Raj, A., Rifkin, S. A., Andersen, E., van Oudenaarden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variability+in+gene+expression+underlies+incomplete+penetrance.">Variability in gene expression underlies incomplete penetrance.</a> Nature. 463, 913-U984 (2010).</li><li>Raj, 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=Detection+of+individual+endogenous+RNA+transcripts+in+situ+using+multiple+singly+labeled+probes.">Detection of individual endogenous RNA transcripts in situ using multiple singly labeled probes.</a> Methods Enzymol. 472 (10), 365-386 (2010).</li><li>Trcek, T., 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=Single-mRNA+counting+using+fluorescent+in+situ+hybridization+in+budding+yeast.">Single-mRNA counting using fluorescent in situ hybridization in budding yeast.</a> Nature Protocols. 7, 408-419 (2012).</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, 877-879 (2008).</li><li>Otsu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Tlreshold+Selection+Method+from+Gray-Level+Histograms.">A Tlreshold Selection Method from Gray-Level Histograms.</a> IEEE Transactions on Systems, Man and Cybernetics. 9, 62-66 (1979).</li><li>Thompson, R. E., Larson, D. R., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+nanometer+localization+analysis+for+individual+fluorescent+probes.">Precise nanometer localization analysis for individual fluorescent probes.</a> Biophys J. 82, 2775-2783 (2002).</li><li>McIsaac, R. 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=Fast-acting+and+nearly+gratuitous+induction+of+gene+expression+and+protein+depletion+in+Saccharomyces+cerevisiae.">Fast-acting and nearly gratuitous induction of gene expression and protein depletion in Saccharomyces cerevisiae.</a> Molecular biology of the cell. 22, 4447-4459 (2011).</li><li>Lubeck, E., Cai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+systems+biology+by+super-resolution+imaging+and+combinatorial+labeling.">Single-cell systems biology by super-resolution imaging and combinatorial labeling.</a> Nature Methods. 9, 743-U159 (2012).</li><li>Levsky, J. M., Shenoy, S. M., Pezo, R. C., 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-cell+gene+expression+profiling.">Single-cell gene expression profiling.</a> Science. 297, 836-840 (2002).</li><li>Wyart, M., Botstein, D., Wingreen, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+Gene+Expression+Dynamics+Using+Pairwise+RNA+FISH+Data.">Evaluating Gene Expression Dynamics Using Pairwise RNA FISH Data.</a> Plos Computational Biology. 6, (2010).</li></ol>

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 border="1">
	<tbody>
		<tr>
			<td>Name of the Reagent</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Vanadyl Ribonucleoside Complex</td>
			<td>NEB</td>
			<td>S1402S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lyticase</td>
			<td>Sigma</td>
			<td>L5263</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>E. coli tRNA</td>
			<td>Roche</td>
			<td>1010954001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BSA (RNase free)</td>
			<td>Ambion</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Beta-mercaptoethanol</td>
			<td>Fisher</td>
			<td>03446l</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DAPI, dilactate</td>
			<td>Sigma</td>
			<td>D9564</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS 10X (RNase free)</td>
			<td>Ambion</td>
			<td>AM9624</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Shelton Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</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>&nbsp;</td>
		</tr>
		<tr>
			<td>Formamide (deionized)</td>
			<td>Ambion</td>
			<td>AM9342</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>Ambion</td>
			<td>AM9932</td>
			<td>&nbsp;</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>&nbsp;</td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</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>&nbsp;</td>
		</tr>
		<tr>
			<td>Polylysine (0.01%)</td>
			<td>Sigma</td>
			<td>P8920</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Warner Instruments</td>
			<td>Cs-18R15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Prolong Gold Mounting Medium</td>
			<td>Invitrogen</td>
			<td>P36934</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>QIAquick Nucleotide Removal Kit</td>
			<td>QIAGEN</td>
			<td>28304</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FISH Probes</td>
			<td>Biosearch Technologies</td>
			<td>&nbsp;</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>&nbsp;</td>
		</tr>
		<tr>
			<td>Cy3, Cy3.5, Cy5 dyes</td>
			<td>GE Healthcare</td>
			<td>&nbsp;</td>
			<td>monofunctional NHS-ester</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</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>&nbsp;</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>U-NSP100V2-SPR, U-NSP101V2-SPR, U-NSP102V2-SPR, U-NSP103V2-SPR,U-NSP104V2-SPR.</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cooled CCD or EMCCD Camera</td>
			<td>Hamamatsu</td>
			<td>C4742-98-24ER</td>
			<td>&nbsp;</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 ...

visualization-analysis-mrna-molecules-using-fluorescence-situ

Research

JoVE Journal

Biology

34.5K vistas.  Princeton University. Este protocolo describe un procedimiento experimental para la realización de fluorescencia In situ La hibridación (FISH) para el recuento de ARNm en células individuales con una resolución de una sola molécula.

Video: 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...

rapid synthesis and screening of chemically activated transcription factors with gfp-based reporters

Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

This protocol describes an experimental procedure for the rapid construction of artificial transcription factors (ATFs) with cognate GFP reporters&#160;and quantification of the ATFs ability to stimulate GFP expression via flow...

a suppressor screen for the characterization of genetic links regulating chronological lifespan in saccharomyces cerevisiae

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Here is a protocol to identify genetic interactions through an increased copy number suppressor screen in Saccharomyces cerevisiae. This method allows researchers to identify, clone, and test suppressors in short-lived yeast mutants. We test the effect of the copy number increase of SIR2 on lifespan in an autophagy null...

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

measurement of mrna decay rates in saccharomyces cerevisiae using rpb1-1 strains

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...

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

visualization of sars-cov-2 using immuno rna-fluorescence in situ hybridization

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

Here, we describe a simple method that combines RNA fluorescence in situ&#160;hybridization (RNA-FISH) with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. This protocol may increase understanding of the molecular characteristics of SARS-CoV-2 RNA-host interactions at a single-cell...

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

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...

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

visualization of bacteria in bladder biopsy sections via fluorescence in situ hybridization

This video demonstrates the visualization of bacteria in deparaffinized bladder tissue from a urinary tract-infected patient. Fluorescently-labeled probe hybridized with bacterial 16s ribosomal RNAs reveals the tissue-associated bacteria and their distribution, providing valuable spatial insights into their density within the...

Visualization of Bacteria in Bladder Biopsy Sections via Fluorescence In Situ Hybridization

Next, place the humidifying chamber into an incubator set to 50 degrees Celsius. Pipette between 50 and 150 microliters of the staining solution directly on top of the tissue so that the rectangle made by the hydrophobic border around the tissue is filled, and close the box gently. Incubate overnight at 50 degrees Celsius in the dark. If the incubator has a window, cover it with aluminum foil to create a dark...

single molecule fluorescence in situ hybridization (smfish) analysis in budding yeast vegetative growth and meiosis

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...

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

quantitative analysis of alternative pre-mrna splicing in mouse brain sections using rna in situ hybridization assay

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

labelling and visualization of mitochondrial genome expression products in baker's yeast saccharomyces cerevisiae

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker&#039;s Yeast Saccharomyces cerevisiae

Baker&#8217;s yeast mitochondrial genome encodes eight polypeptides. The goal of the current protocol is to label all of them and subsequently visualize them as separate...

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

cycloheximide chase analysis of protein degradation in saccharomyces cerevisiae

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Protein abundance reflects the rates of both protein synthesis and protein degradation. This article describes the use of cycloheximide chase followed by western blotting to analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding...

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

microarray analysis for saccharomyces cerevisiae

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...

Microarray Analysis for Saccharomyces cerevisiae

visualization of gut microbiota-host interactions via fluorescence in situ hybridization, lectin staining, and imaging

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

This streamlined protocol details a workflow to detect and image bacteria in complex tissue samples, from fixing the tissue to staining microbes with fluorescent in situ hybridization.

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

visualization of microbiota in tick guts by whole-mount in situ hybridization

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Here, we present a protocol to spatially and temporally assess the presence of viable microbiota in tick guts using a modified whole-mount in situ hybridization...

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

double fluorescence in situ hybridization in fresh brain sections

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate...

Double Fluorescence in situ Hybridization in Fresh Brain Sections

detection of viral rna by fluorescence in situ hybridization (fish)

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking...

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

coupled assays for monitoring protein refolding in saccharomyces cerevisiae

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae.  Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

a fluorescence microscopy assay for monitoring mitophagy in the yeast saccharomyces cerevisiae

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

oral biofilm analysis of palatal expanders by fluorescence in-situ hybridization and confocal laser scanning microscopy

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular...

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

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 process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

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

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

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.

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 ...

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

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

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

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.

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 ...

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 effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

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

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.