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 border="0" cellpadding="0" cellspacing="0" style="width:572px;" width="573">
	<colgroup>
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		<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 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 ...

single-cell-analysis-transcriptionally-active-alleles-single-molecule

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

3.1K Views.  GCC Center for Advanced Microscopy and Image Informatics. 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.

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

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

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

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

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

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

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

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

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve intracellular structures and the dynamics of biomolecules with ~20 nm precision. A prerequisite for SMLM are fluorophores that transition from a dark to a fluorescent state in order to avoid spatio-temporal overlap of their point spread functions in each of the thousands of data acquisition frames. BODIPYs are well-established dyes with numerous conjugates used in conventional microscopy. The transient formation of red-shifted BODIPY ground-state dimers (DII) results in bright single molecule emission enabling single molecule localization microscopy (SMLM). Here we present a simple but versatile protocol for SMLM with conventional BODIPY conjugates in living yeast and mammalian cells. This procedure can be used to acquire super-resolution images and to track single BODIPY-DII states to extract spatio-temporal information of BODIPY conjugates. We apply this procedure to resolve lipid droplets (LDs), fatty acids, and lysosomes in living yeast and mammalian cells at the nanoscopic length scale. Furthermore, we demonstrate the multi-color imaging capability with BODIPY dyes when used in conjunction with other fluorescent&#160;probes. Our representative results show the differential spatial distribution and mobility of BODIPY-fatty acids and neutral lipids in yeast under fed and fasted conditions. This optimized protocol for SMLM can be used with hundreds of commercially available BODIPY conjugates and is a useful resource to study biological processes at the nanoscale far beyond the applications of this work.&#160;

Conventional BODIPY conjugates can be used for live-cell single-molecule localization microscopy (SMLM) through exploitation of their transiently forming, red-shifted ground state dimers. We present an optimized SMLM protocol to track and resolve subcellular neutral lipids and fatty acids in living mammalian and yeast cells at the nanoscopic length scale.

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

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

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

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

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

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately 20 nm to accommodate large tRNA substrates at the A-, P- and E-sites. Consequently, the ribosome dynamics are naturally de-phased quickly. Single molecule method can detect each ribosome separately and distinguish inhomogeneous populations, which is essential to reveal the complicated mechanisms of multi-component systems. We report the details of a smFRET method based on the Nikon Ti2 inverted microscope to probe the ribosome dynamics between the ribosomal protein L27 and tRNAs. The L27 is labeled at its unique Cys 53 position and reconstituted into a ribosome that is engineered to lack L27. The tRNA is labeled at its elbow region. As the tRNA moves to different locations inside the ribosome during the elongation cycle, such as pre- and post- translocation, the FRET efficiencies and dynamics exhibit differences, which have suggested multiple subpopulations. These subpopulations are not detectable by ensemble methods. The TIRF-based smFRET microscope is built on a manual or motorized inverted microscope, with home-built laser illumination. The ribosome samples are purified by ultracentrifugation, loaded into a home-built multi-channel sample cell and then illuminated via an evanescent laser field. The reflection laser spot can be used to achieve feedback control of perfect focus. The fluorescence signals are separated by a motorized filter-turret and collected by two digital CMOS cameras. The intensities are retrieved via the NIS-Elements software.

Single molecule fluorescence energy transfer is a method that tracks the tRNA dynamics during ribosomal protein synthesis. By tracking individual ribosomes, inhomogeneous populations are identified, which shed light on mechanisms. This method can be used to track biological conformational changes in general to reveal dynamic-function relationships in many other complexed biosystems. Single molecule methods can observe non-rate limiting steps and low-populated key intermediates, which are not accessible by conventional ensemble methods due to the average effect.

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

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 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

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 ...

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

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Chapters in this video

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

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

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

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

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

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

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes