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>
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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">
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		<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 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 ...

SM FISH accurately quantifies levels and localization of specific RNase. This method is particularly useful for analyzing cell to cell and allele by allele variation in RNA. Gene transcription is an essential process in cell biology, SM FISH allows for the investigation of RNA content in response to external stimuli at the single cell level and provide spatial information.Following estrogen receptor agonist treatment, wash the cells with cold sterile PBS with calcium and magnesium and fix the cells with 500 microliters of fixation buffer for 30 minutes on ice. At the end of the incubation, wash the cells two times with cold sterile PBS with calcium and magnesium for three minutes before adding 500 microliters of 70%ethanol to each well. Then seal the 24 well plate with a paraffin plastic film and place the plate on a rotator at four degrees Celsius for at least four and up to 16 hours.For RNA FISH hybridization, at the end of the incubation wash the cells one time with 500 microliters of 2X SSC buffer supplemented with 10%formamide for five minutes at room temperature on a rotator. During the incubation, place a piece of parffin plastic film unexposed side up into a glass Petri dish cleaned with an RNA surface decontaminate and place to sterile water-saturated paper towels along the edges of the dish. At the end of the incubation, place evenly spaced 30 microliter droplets of freshly prepared to GREB1 intron and GREB1 exon probe buffer onto the clean side of the paraffin plastic film and use sterile forceps to gently place one cover slip of cells cell side down onto each droplet without bubbles.After sealing the dish with paraffin plastic film, cover the plate with foil and incubate the plate overnight at 37 degrees Celsius on a flat non-rotating surface. The next morning, use forceps to return the cover slips to the 24 well plate cell side up for two 15 minute washes in 500 microliters of fresh 2X SSC buffer supplemented with 10%formamide per wash at 37 degrees Celsius on a heated rotator. After the second wash, counterstain the DNA with one microgram per milliliter of DAPI in 2X SSC buffer for 10 minutes at room temperature on a rotator followed by a five minute wash in 2X SSC buffer at room temperature.Use a paper towel to remove the excess 2X SSC buffer and rinse the samples in nuclease-free water to eliminate excess salts. Then use non-hardening mounting medium to mount the cover slips onto glass slides and seal the cover slips with clear nail polish. For sample imaging, load the slide with the highest expected intensity onto a widefield epifluorescence microscope stage equipped with a 60 or 100 times oil objective in an sCOMS camera and place a droplet of immersion oil onto the objective.For Zstack acquisition, focus on the sample in the DAPI channel and select the top and bottom of the Zstack at the distances at which the DAPI-stained nuclei become out of focus. Use the slide to optimize the amount of illumination from the light source and exposure times for each channel and acquire images from all of the slides under conditions that avoid photo bleaching and or camera pixel saturation. After the acquisition, use 10 cycles of an aggressive restorative algorithm to deconvolve the images and save all of the projection images according to the bit depth of the camera used.For image analysis, first download the Jupiter Notebook from GitHub and install Python Anaconda Distribution version 3.6 or higher. Open the Anaconda prompt and enter the installation command to install SimpleITK for Anaconda. Open the Anaconda Navigator and launch Jupiter Notebook.A web browser will open showing directories and files. Browse through the directory structures to reach the directory containing the downloaded Jupiter Notebook from GitHub. Then open the notebook and press Shift and Enter to run each cell.In this representative experiment, adherent breast cancer cells were treated with an estrogen receptor agonist, or vehicle, for 24 hours. Spectrally separated probe sets against GREB1 intronic and exonic sequences allowed the simultaneous visualization and quantification of nascent and mature mRNA. Importantly, the number of transcriptionally active alleles in each cell that has been shown to be part of the estrogen response time course can be marked.Counting the overlapping intron and exon spots, the E2-treated cells were determined to have a four-fold increased fraction of cells that exhibit two or more active GREB1 alleles compared to vehicle-treated cells. As introns are spliced out of nascent mRNA to produce mature mRNA that consists of only exon sequences, the number of mature mRNA per cell can be counted by quantifying the number of green spots. Plotting these data then allow the shift in distribution of mature GREB1 mRNA cell and the aggregate fold change in the cell population after E2 treatment to be observed.C-FISH allows for investigating multiple RNA species sequentially, MERFISH is a barcoding technique to massively increase multiplexing, and iFISH is used to simultaneously study RNA and proteins.

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JoVE Journal

Biochemistry

3.0K Views.  GCC Center for Advanced Microscopy and Image Informatics. SM FISH accurately quantifies levels and localization of specific RNase. This method is particularly useful for analyzing cell to cell and allele by allele variation in RNA. Gene transcription is an essential process in cell biology, SM FISH allows for the investigation of RNA content in response to external stimuli at the single cell level and provide spatial information.Following estrogen receptor agonist treatment, wash the cells with cold sterile PBS with calcium and magnesium and ...