Name: single cell analysis of transcriptionally active alleles by single molecule fish
Uploaded: 2020-09-20T14:00:00
Description: Watch this Scientific Journal Video about Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH Video at JoVE.com

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 cul...

<ol>
	<li>Russell, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Expression:+Transcription.">Gene Expression: Transcription.</a> iGenetics: A Mendelian Approach. , 331-352 (2006).</li><li>Darnell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variety+in+the+level+of+gene+control+in+eukaryotic+cells.">Variety in the level of gene control in eukaryotic cells.</a> Nature. 297, 365-371 (1982).</li><li>K&#246;hler, A., Hurt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exporting+RNA+from+the+nucleus+to+the+cytoplasm.">Exporting RNA from the nucleus to the cytoplasm.</a> Nature Reviews Molecular Cell Biology. 8, 761-773 (2007).</li><li>Kolodziejczyk, A. A., Kim, J. K., Svensson, V., Marioni, J. C., Teichmann, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Technology+and+Biology+of+Single-Cell+RNA+Sequencing.">The Technology and Biology of Single-Cell RNA Sequencing.</a> Molecular Cell. 58, 610-620 (2015).</li><li>Shah, S., Lubeck, E., Zhou, W., 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=In+situ+Transcription+Profiling+of+Single+Cells+Reveals+Spatial+Organization+of+Cells+in+the+Mouse+Hippocampus.">In situ Transcription Profiling of Single Cells Reveals Spatial Organization of Cells in the Mouse Hippocampus.</a> Neuron. 92, 342-357 (2016).</li><li>Halpern, K. B., 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=Bursty+Gene+Expression+in+the+Intact+Mammalian+Liver.">Bursty Gene Expression in the Intact Mammalian Liver.</a> Molecular Cell. 58, 147-156 (2015).</li><li>Stossi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen-induced+transcription+at+individual+alleles+is+independent+of+receptor+level+and+active+conformation+but+can+be+modulated+by+coactivators+activity.">Estrogen-induced transcription at individual alleles is independent of receptor level and active conformation but can be modulated by coactivators activity.</a> Nucleic Acids Research. 48, 1800-1810 (2020).</li><li>Coassin, S. R., Orjalo, A. V., Semaan, S. J., Johansson, H. E. <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+Nuclear+and+Cytoplasmic+RNA+Variants+Utilizing+Stellaris+RNA+Fluorescence+In+situ+Hybridization+in+Adherent+Cells.">Simultaneous Detection of Nuclear and Cytoplasmic RNA Variants Utilizing Stellaris RNA Fluorescence In situ Hybridization in Adherent Cells.</a> Methods in Molecular Biology. 1211, 189-199 (2014).</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 the Sciences of the United States of America. 63, 378-383 (1969).</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+fluorochrome+labelled+RNA.">A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochrome labelled RNA.</a> Experimental Cell Research. 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+biotinated+nucleotide+analog.">Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with biotinated nucleotide analog.</a> Proceedings of the National Academy of the Sciences of the United States of America. 79, 7331-7335 (1982).</li><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>Raj, A., van den 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>Orjalo, A. V., Johansson, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stellaris+RNA+Fluorescence+In+situ+Hybridization+for+the+Simultaneous+Detection+of+Immature+and+Mature+Long+Noncoding+RNAs+in+Adherent+Cells.">Stellaris RNA Fluorescence In situ Hybridization for the Simultaneous Detection of Immature and Mature Long Noncoding RNAs in Adherent Cells.</a> Methods in Molecular Biology. 1402, 119-134 (2016).</li><li>Johnson, C. V., Singer, R. H., 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=Chapter+3+Fluorescent+Detection+of+Nuclear+RNA+and+DNA:+Implications+for+Genome+Organization.">Chapter 3 Fluorescent Detection of Nuclear RNA and DNA: Implications for Genome Organization.</a> Methods in Cell Biology. 35, 73-99 (1991).</li><li>Levsky, J. M., 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=Fluorescence+in+situ+hybridization:+Past,+present+and+future.">Fluorescence in situ hybridization: Past, present and future.</a> Journal of Cell Science. 116, 2833-2838 (2003).</li><li>Wang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAscope:+A+novel+in+situ+RNA+analysis+platform+for+formalin-fixed,+paraffin-embedded+tissues.">RNAscope: A novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.</a> Journal of Molecular Diagnostics. 14, 22-29 (2012).</li><li>Stossi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+estrogenic+mechanisms+of+bisphenol+A+analogs+through+high+throughput+microscopy-based+contextual+assays.">Defining estrogenic mechanisms of bisphenol A analogs through high throughput microscopy-based contextual assays.</a> Chemistry and Biology. 21, 743-753 (2014).</li><li>Szafran, A. T., Stossi, F., Mancini, M. G., Walker, C. L., Mancini, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+properties+of+non-estrogenic+substituted+bisphenol+analogs+using+high+throughput+microscopy+and+image+analysis.">Characterizing properties of non-estrogenic substituted bisphenol analogs using high throughput microscopy and image analysis.</a> PLoS One. 12, 1-19 (2017).</li><li>Rae, J. M., 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=GREB1+is+a+critical+regulator+of+hormone+dependent+breast+cancer+growth.">GREB1 is a critical regulator of hormone dependent breast cancer growth.</a> Breast Cancer Research and Treatment. 92, 141-149 (2005).</li><li>Heldring, N., 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=Estrogen+receptors:+How+do+they+signal+and+what+are+their+targets.">Estrogen receptors: How do they signal and what are their targets.</a> Physiological Reviews. 87, 905-931 (2007).</li><li>Hah, N., 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=A+rapid,+extensive,+and+transient+transcriptional+response+to+estrogen+signaling+in+breast+cancer+cells.">A rapid, extensive, and transient transcriptional response to estrogen signaling in breast cancer cells.</a> Cell. 145, 622-634 (2011).</li><li>Kadam, P., Bhalerao, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+size+calculation.">Sample size calculation.</a> International Journal of Ayurveda Research. 1, 55-57 (2010).</li><li>Kocanova, 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=Activation+of+estrogen-responsive+genes+does+not+require+their+nuclear+co-localization.">Activation of estrogen-responsive genes does not require their nuclear co-localization.</a> PLoS Genetics. 6, (2010).</li><li>Shaffer, S. M., Wu, M. T., Levesque, M. J., Raj, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turbo+FISH:+A+Method+for+Rapid+Single+Molecule+RNA+FISH.">Turbo FISH: A Method for Rapid Single Molecule RNA FISH.</a> PLoS One. 8, (2013).</li><li>Kim, S. O., Kim, J., Okajima, T., Cho, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+paraformaldehyde-treated+individual+cells+investigated+by+atomic+force+microscopy+and+scanning+ion+conductance+microscopy.">Mechanical properties of paraformaldehyde-treated individual cells investigated by atomic force microscopy and scanning ion conductance microscopy.</a> Nano Convergence. 4, (2017).</li><li>Shieh, T. M., 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=Application+of+ribonucleoside+vanadyl+complex+(RVC)+for+developing+a+multifunctional+tissue+preservative+solution.">Application of ribonucleoside vanadyl complex (RVC) for developing a multifunctional tissue preservative solution.</a> PLoS One. 13, 1-14 (2018).</li><li>Waters, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+and+precision+in+quantitative+fluorescence+microscopy.">Accuracy and precision in quantitative fluorescence microscopy.</a> Journal of Cell Biology. 185, 1135-1148 (2009).</li><li>Vinegoni, C., Feruglio, P. 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 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 border="0" cellpadding="0" cellspacing="0" style="width:572px;" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">17&#946; Estradiol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">E2257</td>
			<td style="width:167px;">CAS Number 50-28-2</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ambion SSC (20X) Buffer, Rnase-free</td>
			<td style="width:71px;">ThermoFisher Scientific</td>
			<td style="width:119px;">AM9770</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Corning DMEM with L-Glutamine and 4.5 g/L Glucose</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">MT10017CV</td>
			<td>Manufactured by Invitrogen</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DAPI</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8417</td>
			<td>Without sodium pyruvate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dextran Sulfate, 50% Solution Sterile</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">3730-100ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s Phosphate Buffered Saline Ca++/Mg++</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">21030CV</td>
			<td>Manufactured by Calbiochem</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethanol, 200 Proof (100%)</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">07-678-005</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Falcon 24-Well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate</td>
			<td style="width:71px;">Corning</td>
			<td align="right" style="width:119px;">353047</td>
			<td>Manufactured by Decon Laboratories</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fetal Bovine Serum (FBS), 500 mL</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">50-753-2978</td>
			<td>Manufactured by Gemini Bio Products</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">GE Healthcare DeltaVision LIVE High Resolution Deconvolution Microscope</td>
			<td style="width:71px;">GE Healthcare</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hyclone Water, Molecular Biology Grade</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">SH3053802</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Immersion Oil 1.516</td>
			<td style="width:71px;">GE Healthcare Life Sciences</td>
			<td align="right" style="width:119px;">29162940</td>
			<td>Manufactured by GE Healthcare Bio-Science</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">L-glutamine Solution</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">MT25005Cl</td>
			<td>Manufactured by Corning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MCF-7 cells</td>
			<td style="width:71px;">ATCC</td>
			<td style="width:119px;">HTB-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Molecular Grade Formamide</td>
			<td style="width:71px;">Promega</td>
			<td style="width:119px;">PAH5051</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Olympus 60x/1.42 NA Objective</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Parafilm</td>
			<td style="width:71px;">Bemis Company, Inc.</td>
			<td style="width:119px;">52858-076</td>
			<td>Distributed by VWR</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Paraformaldehyde, 16% Solution, EM Grade</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:119px;">15710</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Penicilin-Streptomycin Solution</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">MT3001Cl</td>
			<td>Manufactured by Corning</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ribonucleoside Vanadyl Complex</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">101229-716</td>
			<td>Manufactured by New England Biosciences</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNase Away</td>
			<td style="width:71px;">Ambion</td>
			<td align="right" style="width:119px;">9780</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium pyruvate, 100 mM</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">11-360-070</td>
			<td>Manufactured by Gibco</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Thermo Scientific Glass Coverslips, #1.5</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">12-545-81P</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Triton X-100, 100 mL</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">BP151-100</td>
			<td>Manufactured by Fisher BioReagents</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trypsin-EDTA Solution 0.25%</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T4049-500ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vectashield Antifade Mounting Medium 10 mL</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">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 Video at JoVE.com

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH Video (Video) | JoVE

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.

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 ...

Research

JoVE Journal

Biochemistry

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination Video (Video) | JoVE

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers Video (Video) | JoVE

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions Video (Video) | JoVE

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy Video (Video) | JoVE

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level Video (Video) | JoVE

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules Video (Video) | JoVE

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and ...

Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking

Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking Video (Video) | JoVE

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve ...

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

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics Video (Video) | JoVE

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

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis Video (Video) | JoVE

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA Video (Video) | JoVE

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...