MAM, FS and MGM are funded by NIEHS (Project 4, P42ES027704, PI, Rusyn). MAM, FS, RMM, MGM and PKS are supported by the CPRIT-funded GCC Center for Advanced Microscopy and Image Informatics (RP170719). Imaging is also supported by the Integrated Microscopy Core at Baylor College of Medicine with funding from NIH (DK56338, and CA125123), CPRIT (RP150578), the Dan L. Duncan Comprehensive Cancer Center, and the John S. Dunn Gulf Coast Consortium for Chemical Genomics.

To ensure optimal results, the wet lab portion of this protocol requires standard RNase-free precautions at all steps. For example, the use of filtered pipette tips, sterile vessels and RNase-free buffers is highly encouraged. Using RNase inhibitors such as vanadyl ribonucleoside complexes is also suggested, especially for low abundance target RNAs.
1. Cell culture and experimental setup
<ol>
	<li>Maintain adherent MCF-7 breast cancer cells (ATCC #HTB-22) in a T75 tissue culture flask with phenol-red free (only required for hormone stimulation experiments) Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 nM sodium pyruvate, 50 I.U./mL penicillin and 50 &#181;g/mL streptomycin.</li>
	<li>Place acid-etched, poly-D-lysine coated round coverslips (0.16 to 0.19 mm thick, 12 mm diameter) into a 24 multiwell tissue culture plate. 
	NOTE: Use 1 M HCl to acid-etch the coverslips and coat with a 1 mg/mL solution of poly-D-lysine reconstituted in sterile PBS (phosphate-buffered saline without Ca++ and Mg++) according to standard protocols.</li>
	<li>Remove the growth media from the cells and wash once with 5 mL of sterile PBS (phosphate-buffered saline without Ca++ and Mg++). Detach MCF-7 cells using 2-3 mL of Trypsin-EDTA 0.25% solution. Once cells are detached, neutralize the Trypsin-EDTA 0.25% solution with an equal volume of growth media.</li>
	<li>Transfer the cells in the media suspension to a 15 mL conical tube, spin down at 391 x g for 1 minute to pellet out the cells. Carefully remove the media from the conical tube without disturbing the cell pellet and resuspend the cells in an appropriate amount of treatment media. 
	NOTE: Treatment media is phenol-red free DMEM supplemented with 5% charcoal-dextran stripped and dialyzed FBS, 2 mM L-glutamine, 1 nM sodium pyruvate, 50 I.U./mL penicillin and 50 &#181;g/mL streptomycin. This media is essential for steroid hormone stimulation experiments as the serum component is largely depleted of steroids by charcoal stripping the media, and has reduced growth factors via dialysis.</li>
	<li>Seed the cells onto coverslips in the 24 multiwell plate at 60,000 to 70,000 cells per well in 500 &#181;L of treatment media. For this experiment, cell confluency should be around 70-80% at treatment initiation. Optimize cell seeding density depending on cell type, growth rate and length of the experiment to avoid confluency, which complicates image analysis. 
	NOTE: For best imaging and image analysis, avoid cell clumping. For this purpose, mix the aliquot of cells thoroughly by pipetting (or briefly vortex) the cells a few times before dispensing into the wells. Before placing the plated cells back into the incubator, letting the cells settle in the tissue culture hood for about 20 minutes helps reduce cell clumping.</li>
	<li>Incubate cells for at least two days prior to treatments to ensure cell cycle synchronization and background reduction due to any signaling molecules that remain in the stripped/dialyzed serum of growth media.</li>
	<li>After a 48 hour incubation in treatment media, treat MCF-7 cells with 10 nM 17&#946;-estradiol (E2) and vehicle control (DMSO), diluted in treatment media for 24 hours. This treatment uses a saturating dose of E2 to induce a maximal response. Perform time course and dose-response experiments to empirically determine conditions for different target genes, cell models and type of treatments. As an example, see our recent publication7.</li>
</ol>
2. Preparation of buffers
<ol>
	<li>To prepare the fixation buffer, dilute purified, monomeric formaldehyde (sold by electron-microscopy suppliers as 16% paraformaldehyde) to 4% in sterile PBS Ca++/Mg++ (phosphate-buffered saline plus Ca++ and Mg++). Add vanadyl ribonucleoside complexes (VRC) to the fixation buffer for a final concentration of 2 mM to delay RNA degradation.
	<ol>
		<li>Calculate the total amount of fixation buffer based upon requiring 300-500 &#181;L of fixative per well of a 24 multiwell plate. Store the 4% formaldehyde on ice until required in&#160;the fixation step. 
		NOTE: Make the fixation buffer fresh for each experiment. 
		CAUTION: Formaldehyde is a teratogen that is absorbed through the skin. Use this chemical in a fume hood along with appropriate PPE according to your institutional regulations.</li>
	</ol>
	</li>
	<li>For the permeabilization step, prepare 70% ethanol in nuclease free water. An alternative option is 0.5% Triton-X100 in sterile PBS Ca++/Mg++ plus 2 mM of VRC. Calculate the total amount of permeabilization buffer needed by utilizing 500 &#181;L of buffer per well.</li>
	<li>To prepare 9 mL of the hybridization buffer, add 2 mL of stock 50% dextran sulfate, and 1 mL of 20x SSC (saline sodium citrate) buffer to 6 mL of nuclease free water. Vortex to thoroughly mix the hybridization buffer. This buffer can be stored at 4 &#176;C for up to one week.</li>
	<li>There are five separate wash steps that require 2x saline sodium citrate (SSC) buffer. Calculate the final volume of 2x SSC buffer needed by anticipating 500 &#181;L of buffer per coverslip per wash step. Dilute the required amount of stock 20x SSC buffer in nuclease-free water. 2 mM VRC can be added to wash steps as an extra precaution. This buffer can be stored at room temperature for the duration of the processing steps.</li>
</ol>
3. Fixation for RNA FISH
<ol>
	<li>Following treatment, remove the media from the wells and wash once with sterile, cold PBS Ca++/Mg++. If a low adherence cell model is utilized, do not use a vacuum to aspirate liquid; use manual pipetting to carefully remove media from the side of the well and omit the initial wash step. Add 500 &#181;L fixation buffer for 30 minutes on ice. 
	NOTE: If the sample is prone to RNA degradation, use sterile PBS with 2 mM VRC for the wash step at this point.</li>
	<li>Remove fixative and dispose it in chemical waste according to institutional regulations. Wash the cells twice with cold sterile PBS Ca++/Mg++ for 3 minutes.</li>
	<li>Remove PBS and add 500 &#181;L of 70% ethanol to each well. Seal the 24 well plate with a paraffin plastic film. Place on a rotator at 4 &#176;C for a minimum of four hours. It is best to leave the samples in 70% ethanol overnight. The protocol can be paused at this point and the samples can be stored for up to a week in 70% ethanol. 
	NOTE: A 0.5% Triton-X100 in PBS with 2 mM VRC solution may be used as alternative. Incubate the samples in 500 &#181;L of this permeabilization buffer for 20 minutes at room temperature on a rotator. If utilizing this permeabilization buffer, the protocol cannot be paused at this point and must continue through the hybridization step.</li>
</ol>
4. Hybridization for RNA FISH
<ol>
	<li>Remove the permeabilization buffer and wash one time in 500 &#181;L of 2x SSC buffer plus 10% formamide for 5 minutes at room temperature on a rotator.</li>
	<li>Prepare the complete hybridization buffer. Calculate approximately 30 &#181;L of complete hybridization buffer per coverslip. Add formamide to the hybridization buffer to reach the percent needed. For example, if 500 &#181;L of complete hybridization buffer is needed, then it would consist of 450 &#181;L of the previously made hybridization buffer plus 50 &#181;L of molecular grade formamide to reach 10% vol/vol. Vortex briefly to mix. 
	CAUTION: Formamide is a teratogen that is absorbed through the skin. Use this chemical in a fume hood along with appropriate PPE according to institutional regulations.</li>
	<li>Dilute the GREB1 intron (labeled with Atto 647N) and GREB1 exon (labeled with Quasar 570) probes 1:300 in hybridization buffer. Mix via pipetting. Protect this buffer from light and use immediately. 
	NOTE: The probes were designed and manufactured as detailed in Stossi et al7. The probes are usually provided as a dried oligonucleotide probe pool that must be reconstituted in RNAse-free TE buffer according to the manufacturer protocol14. The stock solution for these probes was 12.5 &#956;M, therefore; the working concentration of the probes is 42 nM.</li>
	<li>Prepare a humidifying chamber for the overnight hybridization step. Lay down a piece of paraffin plastic film, unexposed side up, into a glass Petri dish cleaned with a surface decontaminant to remove RNases. Saturate two paper towels with sterile water and place them along the edges of the Petri dish to provide humidity, as drying can destroy specific labeling.</li>
	<li>Aliquot the probes by dotting 30 &#181;L of probes in hybridization buffer onto the clean side of the paraffin plastic film. Distribute the aliquots of probes so that the coverslips do not come into contact with each other.</li>
	<li>Using sterilized forceps, gently flip the coverslips cell side down onto the probes in the glass Petri dish. Avoid air bubbles and do not apply pressure to the coverslips. 
	NOTE: Do not discard the tissue culture plate with the 2x SSC buffer, as it will be needed for subsequent steps.</li>
	<li>Seal the glass Petri dish with paraffin plastic film and cover the plate with foil to prevent light exposure to the fluorophores. Incubate overnight, up to 16 hours, at 37 &#176;C on a flat non-rotating surface. Time of incubation can be varied empirically, however a minimum of 4 hours is recommended. 
	NOTE: The coverslips are susceptible to sliding around when incubating in the hybridization buffer, which can lead to dry spots on the coverslip and damage the sample.</li>
</ol>
5. Preparing samples for imaging 
<ol>
	<li>The next day, remove the coverslips from the humidifying chamber using forceps and return them to the 24 well plate with the cell side up. Add 500 &#181;L of fresh 2x SSC buffer plus 10% formamide to wash out excess probe and non-specific hybridization. 
	NOTE: Protect the samples from light from this point onwards.</li>
	<li>Wash the cells twice with 500 &#181;L of 2x SSC buffer plus 10% formamide for 15 minutes each at 37&#176;C on a heated rotator.</li>
	<li>Counterstain DNA with DAPI at 1 &#181;g/mL in 2x SSC buffer for 10 minutes at room temperature on a rotator.</li>
	<li>Wash one time with 2x SSC buffer for 5 minutes at room temperature.</li>
	<li>Mount the coverslips onto glass slides using non-hardening mounting media after removing any excess 2x SSC buffer with a paper towel and a quick wash in nuclease free water to eliminate excess salts. Finally, seal the coverslips with clear nail polish.</li>
</ol>
6. High resolution microscopy and image processing
<ol>
	<li>Image the samples on a wide-field epifluorescence microscope using a 60x or 100x oil objective and a sCMOS camera. See the table of materials for the specific microscope and objective used for this experiment.
	<ol>
		<li>Complete imaging as soon as the samples are fully processed to avoid time-dependent degradation of the signal. 
		NOTE: Immersion oil with a refractive index (RI) of 1.516 is used in this experiment. Empirical testing should be performed to match the oil RI to specific samples, as RI mismatches will result in aberrations of the point spread function that will affect image deconvolution.</li>
	</ol>
	</li>
	<li>Images are captured with a lateral pixel size of 0.10827 &#181;m.</li>
	<li>Optimize the amount of illumination from the light source (i.e., percent transmittance) and exposure times for each channel (DAPI, TRITC, CY5 filters in this particular example) using the sample that is expected to have the highest intensity (i.e., positive controls). In this experiment, the E2-treated sample is expected to have higher intensity for both GREB1 probe sets. Generally, the percent transmittance for GREB1 intron and exon probes is 50 &#8211; 100%, and the exposure times range from 0.25 &#8211; 0.60 seconds.</li>
	<li>Additionally, acquire images under conditions that avoid photobleaching and/or any saturation of camera pixels. In our experience, there should be a minimum ~ten-fold difference between the background and the signal intensity. Saturation of few pixels/field can occur due to non-specific signals in the sample (i.e., dirt on the coverslip, probe aggregates outside the cell), which can be acceptable, but only if saturated regions are not included in quantitation.</li>
	<li>To set acquisition of a z-stack, focus on the sample in the DAPI channel. Select the top and the bottom of the z-stack at the distances where the DAPI stained nuclei become out of focus. The z-stack step size we used is 0.25 &#181;m per slice and the total z-stack should span the whole cell (about 10 &#181;m in MCF-7 cells). However, the z-stack step size will change according to the objective used and should follow the Nyquist sampling criterion. 
	NOTE: Introns are usually present only in the nucleus; however, exons are both in the nucleus and the cytoplasm. Therefore, setting the z-stack as described above will capture most of the intron and exon labeling as the z-stack encompasses the whole cell.</li>
	<li>Typically, image a minimum of 200 cells for quantitative analysis in preliminary experiments to capture a snapshot of the magnitude of response and variation between cells.</li>
	<li>Some imaged cells are excluded from the final analysis (i.e., drop-out rate) because they will be filtered out by the analysis pipeline (i.e., cells touching the border of the image, apoptotic/mitotic cells). 
	NOTE: When selecting image areas, there are a few factors to keep in mind. Choose areas with evenly spread, non-overlapping cells as defined by DAPI-labeled, nuclear fluorescence. Additionally, try to select areas that have the least number of nuclei along the edges of the image area to avoid counting partial cells. Automated, unbiased imaging is always preferred for experiments requiring statistical analysis.</li>
	<li>After acquisition, deconvolve images using an aggressive restorative algorithm using 10 cycles (i.e., number of iterations). Generate max-intensity projections for image analysis (omit this step if specific 3D analysis is required). Save all projection images according to the bit depth of the camera used; in our case 16-bit TIFF grayscale images were saved. RGB images have a reduced bit depth resulting in a loss of information; therefore, RGB images are not suitable for image analysis.</li>
</ol>
7. Image analysis 
<ol>
	<li>Read the instructions and download the jupyter notebook from github (https://github.com/pankajmath/RNA_FISH_analysis).</li>
	<li>Install Python anaconda distribution (https://www.anaconda.com/distribution/) version 3.6 or higher.</li>
	<li>Open anaconda prompt and type in the installation command to Install Simple ITK for anaconda (https://anaconda.org/SimpleITK/simpleitk).</li>
	<li>Open anaconda navigator and launch jupyter notebook. It will open a web browser showing directories and files. Browse through the directory structures to reach the directory containing the downloaded jupyter notebook from github.</li>
	<li>Open the notebook and run each cell by pressing Shift and Enter simultaneously.</li>
	<li>Follow the details of each function and step provided in the notebook. For a different set of images, one may have to change some parameter values. 
	NOTE: In brief, nuclei are segmented from the DAPI channel using local thresholding with a block size of 251 pixels, holes were filled and objects smaller than 100 pixels removed. Touching nuclei were split using a watershed algorithm. The nuclear mask was then dilated by 100 pixels and watershed was used to separate touching objects using nuclei as basins. To identify steady state RNA (exons), Otsu thresholding was used; for transcriptional active alleles (intron), first a Gaussian blur (sigma=1) was applied and then max entropy thresholding was used.</li>
</ol>

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F., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+dynamic+range+fluorescence+imaging.">High dynamic range fluorescence imaging.</a> IEEE Journal of Selected Topics in Quantum Electronics. 25, (2019).</li><li>Tam, R., Shopland, L. S., Johnson, C. V., McNeil, J. A., Lawrence, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+RNA+FISH+for+visualizing+gene+expression+and+nuclear+architecture.">Application of RNA FISH for visualizing gene expression and nuclear architecture.</a> FISH: A Practical Approach. , 93-117 (2002).</li><li>Jonkman, J., Brown, C. M., Wright, G. D., Anderson, K. I., North, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tutorial:+guidance+for+quantitative+confocal+microscopy.">Tutorial: guidance for quantitative confocal microscopy.</a> Nature Protocols. , (2020).</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>Kwon, S. <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+fluorescence+in+situ+hybridization:+Quantitative+imaging+of+single+RNA+molecules.">Single-molecule fluorescence in situ hybridization: Quantitative imaging of single RNA molecules.</a> BMB Reports. 46, 65-72 (2013).</li><li>Sibarita, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deconvolution+microscopy.">Deconvolution microscopy.</a> Advcances in Biochemical Engineering/Biotechnology. 95, 201-243 (2005).</li><li>Takei, Y., Shah, S., Harvey, S., Qi, L. S., 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=Multiplexed+Dynamic+Imaging+of+Genomic+Loci+by+Combined+CRISPR+Imaging+and+DNA+Sequential+FISH.">Multiplexed Dynamic Imaging of Genomic Loci by Combined CRISPR Imaging and DNA Sequential FISH.</a> Biophysical Journal. 112, 1773-1776 (2017).</li><li>Moffitt, J. R., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+Imaging+with+Multiplexed+Error-Robust+Fluorescence+in+situ+Hybridization+(MERFISH).">RNA Imaging with Multiplexed Error-Robust Fluorescence in situ Hybridization (MERFISH).</a> Methods in Enzymology. 572, 1-49 (2016).</li><li>Shah, 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=Dynamics+and+Spatial+Genomics+of+the+Nascent+Transcriptome+by+Intron+seqFISH.">Dynamics and Spatial Genomics of the Nascent Transcriptome by Intron seqFISH.</a> Cell. 174, 363-376 (2018).</li><li>Xia, C., Babcock, H. P., Moffitt, J. R., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+detection+of+RNA+using+MERFISH+and+branched+DNA+amplification.">Multiplexed detection of RNA using MERFISH and branched DNA amplification.</a> Scientific Reports. 9, 1-13 (2019).</li><li>Lubeck, E., Coskun, A. F., Zhiyentayev, T., Ahmad, M., 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+in+situ+RNA+profiling+by+sequential+hybridization.">Single-cell in situ RNA profiling by sequential hybridization.</a> Nature Methods. 11, 360-361 (2014).</li><li>Wang, G., Moffitt, J. R., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+imaging+of+high-density+libraries+of+RNAs+with+MERFISH+and+expansion+microscopy.">Multiplexed imaging of high-density libraries of RNAs with MERFISH and expansion microscopy.</a> Scientific Reports. 8, (2018).</li><li>Wassie, A. T., Zhao, Y., Boyden, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+microscopy:+principles+and+uses+in+biological+research.">Expansion microscopy: principles and uses in biological research.</a> Nature Methods. 16, 33-41 (2019).</li><li>Zimmerman, S. G., Peters, N. C., Altaras, A. E., Berg, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+RNA+ISH,+RNA+FISH+and+protein-RNA+double+labeling+(IF/FISH)+in+Drosophila+ovaries.">Optimized RNA ISH, RNA FISH and protein-RNA double labeling (IF/FISH) in Drosophila ovaries.</a> Nature Protocols. 8, 2158-2179 (2013).</li><li>Kochan, J., Wawro, M., Kasza, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+detection+of+mRNA+and+protein+in+single+cells+using+immunofluorescence-combined+single-molecule+RNA+FISH.">Simultaneous detection of mRNA and protein in single cells using immunofluorescence-combined single-molecule RNA FISH.</a> Biotechniques. 59, 209-221 (2015).</li></ol>

The authors have nothing to disclose.

The smFISH methodology described is based upon previously-published protocols7,12,14. In this protocol, we explain the critical steps that were optimized from the wet-lab (including seeding density, fixation time, permeabilization, and probe concentration), to imaging and image analysis, providing a full experimental pipeline for laboratories interested in performing single cell analysis of gene transcription.
<p class="jove...

Gene transcription and translation are the two major processes involved in the central dogma of biology, going from the DNA sequence to RNA to protein1. In this study, we focus on the production of messenger RNA (mRNA) during transcription. The nascent RNA molecule includes both non-coding introns and coding exons. Introns are co-transcriptionally spliced out before the mRNA moves into the cytoplasm for translation on the ribosomes2,3.
Traditionally, the measurement of mRNA is performed in bulk populations of cells (hundreds to millions) with classic assays such as RT-qPCR or Northern blotting. While very powerful, these methods are limited as they do not provide insights into cell-to-cell variation (phenotypic heterogeneity), identification of the number of transcriptionally active alleles, and, perhaps more importantly, lack spatial information. More recently, genome wide technologies allowing for single cell RNA sequencing (RNAseq) began to bridge the gap between imaging methods and sequencing4. However, RNAseq suffers from relatively low detection efficiencies and the processing steps result in complete loss of spatial information5. Although single cell RNAseq can provide insights into phenotypic heterogeneity amongst a population of isogenic cells, RNA fluorescence in situ hybridization (RNA-FISH) can facilitate a more complete exploration of target gene expression at the spatial level, and on an allele-by-allele basis6,7.
RNA FISH is a technique that allows for the detection and localization of target RNA molecules in fixed cells. Unlike earlier, laborious methods, current state-of-the-art RNA-FISH utilizes commercially available nucleic acid probes that are complementary to the target RNA sequences, and these probes hybridize to their targets by Watson-Crick base pairing8. The early in situ hybridization techniques involved a similar protocol established by Gall and Pardue in 1969, as a sample was processed with nucleic acid probes that specifically hybridized to a target RNA9. Originally the assay was performed using radioactive or colorimetric detection; however, in the 1980s, the development of fluorescence in situ hybridization (FISH) protocols to DNA FISH and later RNA FISH opened the route towards more sensitive detection, and the potential for multiplexing10,11.
Further developments in RNA FISH have led to the ability to detect and quantify single RNA molecules (smFISH)12,13. Direct detection is a method in which the probes themselves are labeled with fluorophores. A challenge with smFISH is that there is a need for enough hybridizing probes/target to facilitate the detection of fluorescent signals as distinct, diffraction-limited spots. To address this issue, one can use a probe set of short single-stranded DNA oligonucleotides complementary to various regions of the target RNA8,12,14. The binding of multiple probes increases local fluorophore density, making the RNA visible as a distinct, high intensity spot by fluorescence microscopy. This method is advantageous because off-target binding of oligonucleotides in the probe set pool can be distinguishable from the true RNA spot signal by quantitatively analyzing the spot size and intensity to differentiate between true signal and spurious oligonucleotide binding, thus facilitating more accurate analysis by reducing false positives12.
Alternative methods to detect mRNA are the classic BAC clone-based nick translation method and the more recently developed branched DNA system. In the BAC-cloned, nick-translation method, the DNA to be labeled is enzymatically-nicked and a new nucleotide is added to the exposed 3&#8217; end. The activity of DNA polymerase I adds a labeled nucleotide resulting in the desired probe15,16. The branched DNA technique involves oligonucleotide probes that hybridize in pairs to RNA targets and these probes are amplified utilizing multiple signal amplification molecules. This method can significantly improve the signal-to-noise ratio but the amplification can skew accurate quantitation since fluorescent spots can be wildly different in size and intensity17.
As a model system for this protocol, which is fully-employed as part of the NIEHS Superfund Research Program participation to quantify the effects of endocrine disrupting chemicals in Galveston Bay/Houston Ship Channel, we used the estrogen receptor (ER) positive breast cancer cell line MCF-7 treated overnight with an ER agonist, 17&#946;-estradiol (E2)7,18,19. E2 regulates many ER-target genes, including the prototypical ER-target gene, GREB17,20,21,22. The smFISH protocol described here utilizes a set of two spectrally-separated probe sets targeting GREB1; one that hybridizes to exons and the other to introns, allowing for the measurement of both mature and nascent RNA7. We then use epifluorescence microscopy coupled with deconvolution to image smFISH labeled samples, and then apply image analysis routines to quantify the number of active alleles and mature RNAs per cell.&#160;

<table><tbody><tr><td>17&amp;beta; Estradiol</td><td>Sigma-Aldrich</td><td>E2257</td><td>CAS Number 50-28-2</td></tr><tr><td>Ambion SSC (20X) Buffer, Rnase-free</td><td>ThermoFisher Scientific</td><td>AM9770</td><td></td></tr><tr><td>Corning DMEM with L-Glutamine and 4.5 g/L Glucose</td><td>Fisher Scientific</td><td>MT10017CV</td><td>Manufactured by Invitrogen</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D8417</td><td>Without sodium pyruvate</td></tr><tr><td>Dextran Sulfate, 50% Solution Sterile</td><td>Sigma-Aldrich</td><td>3730-100ML</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline Ca++/Mg++</td><td>Corning</td><td>21030CV</td><td>Manufactured by Calbiochem</td></tr><tr><td>Ethanol, 200 Proof (100%)</td><td>Fisher Scientific</td><td>07-678-005</td><td></td></tr><tr><td>Falcon 24-Well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate</td><td>Corning</td><td>353047</td><td>Manufactured by Decon Laboratories</td></tr><tr><td>Fetal Bovine Serum (FBS), 500 mL</td><td>Fisher Scientific</td><td>50-753-2978</td><td>Manufactured by Gemini Bio Products</td></tr><tr><td>GE Healthcare DeltaVision LIVE High Resolution Deconvolution Microscope</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>Hyclone Water, Molecular Biology Grade</td><td>Fisher Scientific</td><td>SH3053802</td><td></td></tr><tr><td>Immersion Oil 1.516</td><td>GE Healthcare Life Sciences</td><td>29162940</td><td>Manufactured by GE Healthcare Bio-Science</td></tr><tr><td>L-glutamine Solution</td><td>Fisher Scientific</td><td>MT25005Cl</td><td>Manufactured by Corning</td></tr><tr><td>MCF-7 cells</td><td>ATCC</td><td>HTB-22</td><td></td></tr><tr><td>Molecular Grade Formamide</td><td>Promega</td><td>PAH5051</td><td></td></tr><tr><td>Olympus 60x/1.42 NA Objective</td><td>Olympus</td><td></td><td></td></tr><tr><td>Parafilm</td><td>Bemis Company, Inc.</td><td>52858-076</td><td>Distributed by VWR</td></tr><tr><td>Paraformaldehyde, 16% Solution, EM Grade</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Penicilin-Streptomycin Solution</td><td>Fisher Scientific</td><td>MT3001Cl</td><td>Manufactured by Corning</td></tr><tr><td>Ribonucleoside Vanadyl Complex</td><td>VWR</td><td>101229-716</td><td>Manufactured by New England Biosciences</td></tr><tr><td>RNase Away</td><td>Ambion</td><td>9780</td><td></td></tr><tr><td>Sodium pyruvate, 100 mM</td><td>Fisher Scientific</td><td>11-360-070</td><td>Manufactured by Gibco</td></tr><tr><td>Thermo Scientific Glass Coverslips, #1.5</td><td>Fisher Scientific</td><td>12-545-81P</td><td></td></tr><tr><td>Triton X-100, 100 mL</td><td>Fisher Scientific</td><td>BP151-100</td><td>Manufactured by Fisher BioReagents</td></tr><tr><td>Trypsin-EDTA Solution 0.25%</td><td>Sigma-Aldrich</td><td>T4049-500ML</td><td></td></tr><tr><td>Vectashield Antifade Mounting Medium 10 mL</td><td>Fisher Scientific</td><td>NC9265087</td><td>Manufactured by Vector Laboratories</td></tr></tbody></table>

single cell analysis of transcriptionally active alleles by single molecule fish

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

To analyze hormonal responses via smFISH, as an example, we chose the estrogen receptor (ER) model used in high throughput assays to determine the presence of endocrine disrupting chemicals in environmental disasters in the context of participation in the NIEHS Superfund Research Program7. In this experiment, adherent MCF-7 breast cancer cells were treated with the ER agonist 17&#946;-estradiol (E2, 10 nM) or vehicle (DMSO) for 24 hours. Spectrally-separated probe sets against GREB1 intronic (Atto...

Watch this Scientific Journal Video about Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH at JoVE.com

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

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Research

JoVE Journal

Biochemistry

3.1K Views.  GCC Center for Advanced Microscopy and Image Informatics. Single molecule RNA fluorescentie in situ hybridisatie (smFISH) is een methode om niveaus en lokalisatie van specifieke RNA's op het niveau van één cel nauwkeurig te kwantificeren. Hier rapporteren we onze gevalideerde laboratoriumprotocollen voor natte-bench verwerking, beeldvorming en beeldanalyse voor eencellige kwantificering van specifieke RNA's.

Video: Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

Genetics

Transcriptome Analysis of Single Cells

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in culture.1,2,3 In the case of the brain, regions such as the hippocampus contain a complex arrangement of different cell types, each with distinct mRNA profiles. The ability to harvest single cells allows for a more in depth investigation into the molecular differences between and within cell populations. We describe a simple and rapid method for harvesting cells for further processing. Pipettes often used in electrophysiology are utilized to isolate (using aspiration) a cell of interest and conveniently deposit it into an Eppendorf tube for further processing with any number of molecular biology techniques. Our protocol can be modified for the harvest of dendrites from cell culture or even individual cells from acute slices.
We also describe the aRNA amplification method as a major downstream application of single cell isolations. This method was developed previously by our lab as an alternative to other gene expression analysis techniques such as reverse-transcription or real-time polymerase chain reaction (PCR).4,5,6,7,8 This technique provides for linear amplification of the polyadenylated RNA beginning with only femtograms of material and resulting in microgram amounts of antisense RNA. The linearly amplified material provides a more accurate estimation than PCR exponential amplification of the relative abundance of components of the transcriptome of the isolated cell. The basic procedure consists of two rounds of amplification. Briefly, a T7 RNA polymerase promoter site is incorporated into double stranded cDNA created from the mRNA transcripts. An overnight in vitro transcription (IVT) reaction is then performed in which T7 RNA polymerase produces many antisense transcripts from the double stranded cDNA. The second round repeats this process but with some technical differences since the starting material is antisense RNA. It is standard to repeat the second round, resulting in three rounds of amplification. Often, the third round in vitro transcription reaction is performed using biotinylated nucleoside triphosphates so that the antisense RNA produced can be hybridized and detected on a microarray.7,8

In this article we describe a simple method for the harvesting of single cells from rat primary neuronal cultures and subsequent transcriptome analysis using aRNA amplification. This approach is generalizable to any cell type.

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in ...

Neuroscience

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 ...

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is inherently a single-cell assay, however prior to molecular analysis, the target cells are often prospectively isolated in bulk, thereby losing single-cell resolution. Single-cell gene expression analysis provides a means to understand molecular differences between individual cells in heterogeneous cell populations. In bulk cell analysis an overrepresentation of a distinct cell type results in biases and occlusions of signals from rare cells with biological importance. By utilizing FACS index sorting coupled to single-cell gene expression analysis, populations can be investigated without the loss of single-cell resolution while cells with intermediate cell surface marker expression are also captured, enabling evaluation of the relevance of continuous surface marker expression. Here, we describe an approach that combines single-cell reverse transcription quantitative PCR (RT-qPCR) and FACS index sorting to simultaneously characterize the molecular and immunophenotypic heterogeneity within cell populations.
In contrast to single-cell RNA sequencing methods, the use of qPCR with specific target amplification allows for robust measurements of low-abundance transcripts with fewer dropouts, while it is not confounded by issues related to cell-to-cell variations in read depth. Moreover, by directly index-sorting single-cells into lysis buffer this method, allows for cDNA synthesis and specific target pre-amplification to be performed in one step as well as for correlation of subsequently derived molecular signatures with cell surface marker expression. The described approach has been developed to investigate hematopoietic single-cells, but have also been used successfully on other cell types.
In conclusion, the approach described herein allows for sensitive measurement of mRNA expression for a panel of pre-selected genes with the possibility to develop protocols for subsequent prospective isolation of molecularly distinct subpopulations.

Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations.

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

Transcriptional Analysis by Nascent RNA FISH of In Vivo Trophoblast Giant Cells or In Vitro Short-term Cultures of Ectoplacental Cone Explants

The placenta derives from one extra-embryonic lineage, the trophectoderm. In the peri-implantation murine blastocyst, mural trophectoderm cells differentiate into primary trophoblast giant cells (TGCs) while the polar trophectoderm overlying the inner cell mass continues to proliferate later differentiating into secondary TGCs. TGCs play a key role in developing placenta and are essential for a successful pregnancy. Investigation of transcriptional regulation of specific genes during post-implantation development can give insights into TGCs development. Cells of the ectoplacental cone (EPC) from embryos at 7-7.5 days of gestation (E7-7.5), derived from the polar trophectoderm, differentiate into secondary TGCs1. TGCs can be studied in situ, on cryostat sections of embryos at E7 although the number of TGCs is very low at this stage. An alternative means of analyzing secondary TGCs is to use short-term cultures of individual EPCs from E7 embryos. We propose a technique to investigate the transcriptional status of genes of interest both in vivo and in vitro at the single-cell level using fluorescent in situ hybridization (RNA FISH) to visualize nascent transcripts. This technique provides a direct readout of gene expression and enables assessment of the chromosomal status of TGCs, which are large endoreplicating cells. Indeed, a key feature of terminal differentiation of TGCs is that they exit the cell cycle and undergo multiple rounds of endoreplication.This approach can be applied to detect expression of any gene expressed from autosomes and/or sex chromosomes and can provide important information into developmental mechanisms as well as placental diseases.

Transcriptional  Analysis by Nascent RNA FISH of In Vivo Trophoblast Giant Cells or In Vitro Short-term Cultures of Ectoplacental Cone Explants

Trophoblast giant cells (TGCs) play a key role in the placenta to ensure a healthy pregnancy. We present a protocol for assessing the transcriptional status of genes in TGCs by nascent fluorescent in situ hybridization on cryostat sections of post-implantation embryos or short-term cultures of embryonic day 7 ectoplacental cones.

The placenta derives from one extra-embryonic lineage, the trophectoderm. In the peri-implantation murine blastocyst, mural trophectoderm cells ...

Developmental Biology

3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells

A major question in cell biology is genomic organization within the nuclear space and how chromatin architecture can influence processes such as gene expression, cell identity and differentiation. Many approaches developed to study the 3D architecture of the genome can be divided into two complementary categories: chromosome conformation capture based technologies (C-technologies) and imaging. While the former is based on capturing the chromosome conformation and proximal DNA interactions in a population of fixed cells, the latter, based on DNA fluorescence in situ hybridization (FISH) on 3D-preserved nuclei, allows contemporary visualization of multiple loci at a single cell level (multicolor), examining their interactions and distribution within the nucleus (3D multicolor DNA FISH). The technique of 3D multicolor DNA FISH has a limitation of visualizing only a few predetermined loci, not permitting a comprehensive analysis of the nuclear architecture. However, given the robustness of its results, 3D multicolor DNA FISH in combination with 3D-microscopy and image reconstruction is a possible method to validate C-technology based results and to unambiguously study the position and organization of specific loci at a single cell level. Here, we propose a step by step method of 3D multicolor DNA FISH suitable for a wide range of human primary cells and discuss all the practical actions, crucial steps, notions of 3D imaging and data analysis needed to obtained a successful and informative 3D multicolor DNA FISH within different biological contexts.

3D multicolor DNA FISH represents a tool to visualize multiple genomic loci within 3D preserved nuclei, unambiguously defining their reciprocal interactions and localization within the nuclear space at a single cell level. Here, a step by step protocol is described for a wide spectrum of human primary cells.

A major question in cell biology is genomic organization within the nuclear space and how chromatin architecture can influence processes such as gene ...

Single Nucleotide Polymorphism-sensitive FISH Detection of Locus-specific Ribosomal RNA Transcription in Drosophila melanogaster

In order to ensure sufficient ribosomal RNA (rRNA) is transcribed for ribosome function, genomes contain hundreds of tandem duplications of the sequences that encode rRNAs, composing regions called ribosomal DNA (rDNA) loci. Many organisms' genomes contain more than one rDNA locus distributed across different chromosomes, and rRNA may be transcribed from multiple rDNA loci or only a single locus. Changes in rRNA transcription sources often indicate ribosomal distress. However, the homogeneity of rRNA obstructs distinguishing if rRNA is transcribed from multiple or a single rDNA locus. Here, we describe a method that uses single nucleotide polymorphism-sensitive fluorescence in situ hybridization (SNP-FISH) to distinguish rRNA transcription between Drosophila melanogaster rDNA loci on the X and Y chromosomes. This method leverages locus-specific rRNA variants to enable easy detection of the source of rRNA transcription with single-cell resolution. This assay can be applied to any Drosophila cell type and may be adapted for use in other systems.

The protocol describes single nucleotide polymorphisms-sensitive fluorescence in situ hybridization (SNP-FISH) method to distinguish ribosomal RNA transcripts derived from the X or Y chromosome ribosomal DNA locus in Drosophila melanogaster.

In order to ensure sufficient ribosomal RNA (rRNA) is transcribed for ribosome function, genomes contain hundreds of tandem duplications of the sequences ...

Detection and Quantification of Single mRNA Molecules in Drosophila

Studying Muscle Transcriptional Dynamics at Single-molecule Scales in Drosophila

Skeletal muscles are large syncytia made up of many bundled myofibers that produce forces and enable body motion. Drosophila is a classical model to study muscle biology. The combination of both Drosophila genetics and advanced omics approaches led to the identification of key conserved molecules that regulate muscle morphogenesis and regeneration. However, the transcriptional dynamics of these molecules and the spatial distribution of their messenger RNA within the syncytia cannot be assessed by conventional methods. Here we optimized an existing single-molecule RNA fluorescence in situ hybridization (smFISH) method to enable the detection and quantification of individual mRNA molecules within adult flight muscles and their muscle stem cells. As a proof of concept, we have analyzed the mRNA expression and distribution of two evolutionary conserved transcription factors, Mef2 and Zfh1/Zeb. We show that this method can efficiently detect and quantify single mRNA molecules for both transcripts in the muscle precursor cells, adult muscles, and muscle stem cells.

Author Spotlight: Investigating mRNA Spatial Distribution in Drosophila Muscle Tissue 

Drosophila is a well-established model for studying key molecules that regulate myogenesis. However, current methods are insufficient to determine mRNA transcriptional dynamics and spatial distribution within syncytia. To address this limitation, we optimized an RNA fluorescence in situ hybridization method allowing the detection and quantification of mRNAs at a single-molecule scale.

Skeletal muscles are large syncytia made up of many bundled myofibers that produce forces and enable body motion. Drosophila is a classical model to study ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Biology

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.

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.

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

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

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