We thank Dr. Daniel R. Larson for his generous help with the installation and use of the Spot Finding and Tracking Program&#160;13&#160;and the technical support of the University of Nebraska Lincoln Microscopy Core for the confocal microscopy imaging. This study represents a contribution of the University of Nebraska Agricultural Research Division, Lincoln, Nebraska and was supported by UNL Hatch Funds (NEB-26-206/Accession number -232435 and NEB-26-231/Accession number -1013511).

Animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Nebraska-Lincoln and all methods were performed in accordance with relevant guidelines and regulations. For this study, CD-1 outbred mice had ad libitum access to normal rodent chow and water; they were maintained in a 12:12 dark: light cycle.
1. Preparation of required media
<ol>
	<li>For base media (OMM), add 100 mM NaCl, 5 mM KCl, 0.5 mM KH2PO4, and 1.7 mM CaCl2-2H2O to 100 mL sterile water. 
	NOTE: OMM medium can be stored for up to 1 month.</li>
	<li>For complete media (OMOPS), add 20 mM 3-morpholinopropane-1-sulfonic acid (MOPS), 1.2 mM MgSO4-7H2O, 0.5 mM glucose, 6 mM L-lactate, 1 mM ala-gln, 0.1 mM taurine, 1x non-essential amino acids (NEAA), 0.01 mM ethylenediaminetetraacetic acid (EDTA), 10 &#181;M alpha lipoic acid, 10 &#181;g/mL undiluted gentamicin, 21 mM 1 M NaOH, 5 mM NaHCO3, 0.2 mM Pyruvate, 0.5 mM Citrate, 4 mg/mL FAF BSA to a 1:10 dilution of OMM in sterile water for a total volume of 100 mL. Sterilize the medium with a 0.22 &#181;M filter. 
	NOTE: OMOPS can be stored for up to 1 week.</li>
	<li>For the holding medium (HM) add 5% fetal bovine serum to OMOPS. Make 2 mL HM per mouse.</li>
	<li>For hyaluronidase solution add 0.1 mg/mL of hyaluronidase derived from bovine testes, to 1 mL HM.</li>
	<li>For the fixation buffer combine 4% paraformaldehyde in 10 mL of 1x PBS along with 0.1% embryo grade polyvinylpyrrolidone (PVP)14.</li>
	<li>To prepare 50 mL wash buffer (WB), add 0.1% non-ionic surfactant and 0.1% PVP to 1x PBS14.</li>
	<li>To Prepare 10 mL permeabilization buffer, add 1% non- ionic surfactant to 1x PBS14. 
	NOTE: The wash and permeabilization buffers described above replace the proprietary buffers in the commercially available kits.</li>
</ol>
2. Collection of ovulated oocytes from female mice
<ol>
	<li>Preparation:
	<ol>
		<li>Stimulate female mice at 5-8 weeks of age by intraperitoneal (IP) injection of 5 IU equine chorionic gonadotropin (eCG) followed by 5 IU human chorionic gonadotropin (hCG) 44-48 h later15,16.</li>
		<li>Keep 35 mm Petri dishes containing 2 mL of HM on a 37 &#176;C warming plate. Pipette one, 100-&#181;L drop of HM containing diluted hyaluronidase followed by three, 50 &#181;L drops of HM without hyaluronidase in a 60 mm petri dish. Place plates containing drops on the 37 &#176;C warming plate prior to use. 
		NOTE: The hyaluronidase drops should be made just prior to the dissection of each oviduct pair to prevent the evaporation and concentration of the components of HM with or without hyaluronidase.</li>
	</ol>
	</li>
	<li>Euthanize mice, 16 h after the IP injection of hCG, using isoflurane overdose followed by cervical dislocation.</li>
	<li>Clean the mouse using 70% ethanol. Expose the abdominal cavity and visualize the female reproductive tract. Hold the ovary with forceps and remove the uterine ligaments and excess adipose tissue from around the ovary. Cut the oviduct from the uterus and place the ovary-oviduct pair in the warm HM in the 35 mm dish.</li>
	<li>Remove the ovary and any surrounding adipose tissue. Tear the swollen ampulla of the oviduct using a &#189; inch 27-gauge needle. Push the oviduct at the site of the tear and the cumulus cell-oocyte complexes (COCs) will be expelled. Transfer the ovulated oocytes, which are presumed to be in metaphase II (MII) of meiosis, to the 100 &#956;L drop containing HM media with hyaluronidase using a mouth pipette (Figure 2).</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59414/59414fig2.jpg" /> 
Figure 2: Parts of the mouth pipettor used to transfer oocytes. (A) mouth piece (B) 0.22 um, 4mm filter (C) aspirator tubing (D) 1000 &#956;L pipet tip (E) 9&#34; Pasteur pipet. <a href="https://www.jove.com/files/ftp_upload/59414/59414fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Pipette the MII oocyte-cumulus cell complexes up and down in the hyaluronidase containing HM with the mouth pipette to dislodge cumulus cells. Transfer each oocyte, once they are devoid of cumulus cells to a wash drop containing HM only using the mouth pipette. Repeat this for each wash droplet. Do not transfer fragmented or transparent oocytes15. 
	NOTE: It is important to transfer the oocytes from each drop in the 35 mm dish with as little HM as possible. This is true for every transfer in the protocol. The MII oocytes must not remain in the hyaluronidase containing HM medium for more than one minute.</li>
</ol>
3. SM-FISH Staining of Oocytes
<ol>
	<li>Fix oocytes in an individual well of a 6-well plate containing 500 &#181;L of Fixation Buffer. Submerge 20 oocytes or less in the well. Incubate for 20 min at room temperature. 
	NOTE: Each SM-FISH staining step occurs within an individual well in a 6-well conical plate. Ensure that oocytes are completely submerged in buffers and not floating on top of the buffer. Each step should be performed with 20 oocytes or less in each well.</li>
	<li>Transfer fixed oocytes to 500 &#956;L of wash buffer (WB described in step 1.6) for 10 min each. Repeat 2 more times.</li>
	<li>Incubate oocytes in permeabilization buffer for 30 min at room temperature. 
	NOTE: The permeabilization buffer described in step 1.7 replaces the propriety permeabilization buffer.
	<ol>
		<li>Gather probe sets and quickly spin them down in a microcentrifuge. Warm each probe set for 10 min in a 40 &#176;C water bath or incubator. Cool to the room temperature. 
		NOTE: This step should be performed during the permeabilization incubation</li>
	</ol>
	</li>
	<li>Wash oocytes in 500 &#181;L of WB for 10 min at room temperature.</li>
	<li>Transfer oocytes to 80 &#956;L of Protease III Buffer (available from the kit), that is diluted 1:8 in 1X PBS, for 30 min at room temperature. 
	NOTE: The 80 &#181;L volume adequately covers the bottom of an individual well in a 6-well plate.</li>
	<li>Wash oocytes in 500 &#181;L of WB for 10 min at room temperature.</li>
	<li>Dilute the warmed probe sets for Nanog, Pou5f1, and DapB (a negative control gene), 1:50 in probe diluent. Incubate oocytes in 80 &#956;L of the transcript-specific probe for 2 hours at 40&#176; C. 
	NOTE: Each proprietary probe set is available in one of three fluorescence channels (C1, C2, and C3). The Nanog and Pou5f1 probes were tagged with C2 and C3, respectively.</li>
	<li>Warm the proprietary, Amplifier 1 (AMP 1), Amplifier 2 (AMP2), Amplifier 3 (AMP3) and Amplifier 4-fluorescence (AMP 4-FL) at room temperature. 
	NOTE: This step should be performed during the 2-hour transcript-specific probe incubation.</li>
	<li>Transfer the oocytes to 500 &#956;L of WB and incubate for 10 min at room temperature.</li>
	<li>Incubate oocytes sequentially in amplification buffers.
	<ol>
		<li>Incubate oocytes in 80 &#181;L of AMP1 for 30 min at 40&#176; C. Transfer oocytes to 500 &#181;L of WB for 10 min at room temperature.</li>
		<li>Incubate oocytes in 80 &#181;L of AMP2 for 15 min at 40 &#176;C. Transfer oocytes to 500 &#181;L of WB for 10 min at room temperature.</li>
		<li>Incubate the oocytes in 80 &#181;L of AMP3 for 30 min at 40 &#176;C. Transfer oocytes to 500 &#181;L of WB for 10 min at room temperature. 
		NOTE: The remainder of the protocol is performed in the dark because AMP-FL contains the fluorophore. When working under the dissecting microscope, reduce the light as much as possible.</li>
		<li>Add oocytes to 80 &#956;L of AMP4-FL for 15 min at 40&#176; C. 
		NOTE: AMP4-FL is provided as alternative buffer-A (Alt-A), Alt-B or Alt-C. Select the AMP4-FL buffer dependent on which emission wavelength is desired.</li>
	</ol>
	</li>
	<li>Wash oocytes in 500 &#181;L of WB for 10 min at room temperature. Incubate oocytes in 80 &#181;L of DAPI for 20 min at room temperature. Wash oocytes in 500 &#181;L of WB for 5 min at room temperature.</li>
	<li>Pipette 12 &#181;L of anti-fading mounting media onto the center of a slide without adding bubbles to the reagent. Transfer oocytes with as little WB as possible into the mounting media and apply a coverslip.
	<ol>
		<li>Tilt the coverslip at an angle and slowly and gently place over the liquid on the slide. Avoid pressing the coverslip too hard to prevent distortion of the oocytes and introduction of bubbles.</li>
		<li>Store the slides in a dark box to dry overnight at room temperature. Coat the edges of the slides in clear nail polish to seal coverslip.</li>
	</ol>
	</li>
	<li>Use a standard microscope to find oocytes on the slide and circle with a permanent marker. 
	NOTE: This step is not required but improves locating oocytes on the slide. For best results, image slides within 1 to 5 days as the fluorescent signal will begin to fade.</li>
</ol>
4. Image Processing
<ol>
	<li>Image the 3-dimensional oocytes, using z step confocal microscopy. 
	NOTE: To accurately analyze the images, each z step should be 1.0 &#181;m/slice.</li>
	<li>Save confocal images as a compressed nd2 or individual .TIFF files for each oocyte. Both image types are compatible with the open source image processing program, Fiji.</li>
	<li>Download and install the open access Fiji software (https://imagej.net/Fiji/Downloads).&#160;
	<ol>
		<li>Drag nd2 files into Fiji and choose hyperstack. If confocal images were saved as .TIFF files skip to step 4.4. 
		NOTE: When the nd2 file is dropped into Fiji the hyperstack dropdown should appear automatically.</li>
		<li>Click the Image tab, Select Color, and click Split Channels to separate the fluorescent channels of the nd2 file.</li>
		<li>Generate individual .TIFF files for each z-slice of the oocyte in each fluorescent channel. Click the Image tab, select Stacks, and click Stack to Images. Click the Image tab, select Type, and click RGB Color to convert each z slice to an individual RGB color image. 
		NOTE: The RGB color is artificial and can be chosen as desired for each emission wavelength.</li>
		<li>Save each converted image as .TIFF file. Place images from a single oocyte for each fluorescent channel in a new folder to avoid confusion during stitching (step 4.3).</li>
	</ol>
	</li>
	<li>Normalize each .TIFF image for Pou5f1 and Nanog using negative control images (DapB). 
	NOTE: Normalization is performed using a photo editing program. Make sure to remove the same levels of background fluorescence from each control image.</li>
	<li>Open each normalized .TIFF file in Fiji to stitch together all z-slices for each oocyte in each wavelength.
	<ol>
		<li>Click the Plugins tab, select Stitching, and click on Grid/Collection (Figure 3A). Select Sequential Images from the drop-down menu and click OK (Figure 3B).</li>
		<li>Browse the directory and select the folder containing all of the z-slice images for an individual oocyte at one wavelength (see step 4.3.4). Click OK.</li>
		<li>Move the slider at the bottom of the stitched image to the appropriate color channel for the wavelength used and create the final RGB stitched image by clicking Image, selecting Type, and click RGB Color. 
		NOTE: This image will be used for fluorescence quantitation described in step 4.6 below.</li>
	</ol>
	</li>
	<li>Convert the stitched image to 32-bit maximum projected picture. Click Image, select Type, and click 32-bit (Figure 3C). Save this image as a new .TIFF file.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59414/59414fig3.jpg" /> 
Figure 3: Stitching together of confocal z-series images of oocytes. (A) Screenshot showing the Plug-in Grid/Collection tool in Fiji that was used to produce composite images of the oocyte. (B) Sequential Images uses fluorescence overlap between sequential .TIFF files to generate a composite image. (C) The composite image was saved as a 32-bit .TIFF file. <a href="https://www.jove.com/files/ftp_upload/59414/59414fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="7">
	<li>Download and install the Spot Finding and Tracking Program13, which is available from the website for D.R. Larson, an investigator at the National Institutes of Health National Cancer Institute (https://ccr.cancer.gov/Laboratory-of-Receptor-Biology-and-Gene-Expression/daniel-r-larson). Download and install the open access virtual machine for the interactive data language (IDL) operating system which is required to run the Spot Finding and Tracking Program (http://www.spacewx.com/pdf/idlvm.pdf).&#160;&#160;</li>
	<li>Open the 32-bit, stitched image, which was generated in step 4.6 (Figure 4A), in the Spot Finding and Tracking Program. Select the Localize dropdown and click Localize (Figure 4B), which will calculate the number spots found in the image. 
	NOTE: Each spot counted represents an individual mRNA. Band pass and photon threshold settings are shown in the screenshot (Figure 4B). For this protocol, default for each threshold setting was used. Representative positive and background spots are shown (Figure 4C).</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59414/59414fig4.jpg" /> 
Figure 4: Quantification of mRNAs using Spot Finder and Tracking. (A) Individual z-series images were stitched together as described in Figure 3 and saved as a 32-bit maximum projected .TIFF file. (B) Composite image was opened in Spot Finder and Tracking. Localize was used to count the fluorescent spots (red box). Band pass and photon threshold are indicated by the blue box. (C) The blue arrow points to a positive signal (above threshold). The white arrow shows a fluorescent spot below the threshold and, therefore, not counted. <a href="https://www.jove.com/files/ftp_upload/59414/59414fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors have nothing to declare

A series of minor steps during the protocol will ensure successful fluorescence and accurate counts of mRNAs. First, the protocol must be performed immediately after collection and fixation of the oocytes. Note that PVP is added to the 4% paraformaldehyde fixation buffer to prevent oocytes from sticking to each other. We found that it is necessary to perform the experiment immediately after the collection and fixation of the oocytes. Any delay results in a much lower fluorescence signal that would result in undercounting...

Reverse-transcriptase polymerase chain reaction (PCR) has been the gold standard for mRNA quantitation. Two assays, digital PCR (dPCR)1 and quantitative, real time PCR (qPCR)2 are currently used. Of the two PCR techniques, dPCR has greater sensitivity than qPCR suggesting that it could be used to measure mRNA abundance in single cells. However, in our hands, dPCR analysis of low abundance mRNAs in pools of 5 to 10 oocytes per each experimental sample has produced data with low reproducibility and high variation3. This is likely due to the experimental error associated with RNA extraction and reverse transcription efficiency. RNA sequencing has also been performed using a single mouse and human oocytes4,5. This technique requires cDNA amplification steps required for the library generation which likely increases variability within an experimental group. Furthermore, low abundance transcripts may not be detectable. Although sequencing prices have gone down&#160;the last few years, it can still be cost prohibitive due to the high cost of bioinformatics analyses. Finally, mRNA localization is a dynamic process with spatial changes contributing to protein function6. Therefore, we set out to adopt a technique that would produce accurate and reproducible quantitative measures and localization of individual mRNAs in single oocytes.
Branched DNA coupled to fluorescence in situ hybridization amplifies fluorescence signal rather than amplifying RNA/cDNA enabling detection of single mRNAs in individual cells 7,8,9. The assay is performed through a series of hybridization, amplification (using branched DNA), and fluorescence labeling steps in order to amplify the fluorescence signal7. The technique begins with binding of 18- to 25-base oligonucleotide probe pairs that are complementary to a specific mRNA3,8,10. Fifteen to twenty probe pairs are designed for each transcript ensuring specificity for the target transcript. The mRNA-specific hybridization is followed by pre-amplifier and amplifier probes that form a branched configuration. Approximately, 400 label fluorophores bind to each amplifier, resulting in an 8000-fold increase in fluorescence allowing detection of individual mRNAs (Figure 1)11.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59414/59414fig1.jpg" /> 
Figure 1: Schematic of the SM-FISH protocol. Sequential hybridization of transcript specific probe, branched DNA amplifier and fluorophore to a target mRNA is shown. <a href="https://www.jove.com/files/ftp_upload/59414/59414fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Previous studies using single molecule fluorescence in situ hybridization (SM-FISH) localized &#946;-actin mRNAs in individual neurons12 and human papillomavirus DNA in cervical cancer cell lines7. The computer software Spot Finding and Tracking Program identifies individual punctate fluorescent signal and has been successfully used to quantify the number of mRNAs in each cell3,13.
Based on the results of mRNA detection in neurons12, we hypothesized that SM-FISH would prove a useful tool to quantitate transcript levels in murine oocytes and embryos including low abundance mRNAs. However, the technique is optimized for the use with adherent fixed cells and formaldehyde fixed paraffin embedded (FFPE) tissue sections. Oocytes cannot adhere to a slide, even when they are coated with Poly-L-lysine. Furthermore, they are more fragile than somatic cells and tissue sections resulting in cell lysis when subjected to some of the proprietary buffers in commercially available kits3. To overcome these challenges, oocytes were fixed and manually transferred between drops of the buffers. Furthermore, permeabilization and wash buffers in the kits were replaced to reduce the cell lysis. Predesigned probes are purchased alongside the FISH kit or specific transcripts can be requested. Each proprietary probe set is available in one of three fluorescence channels (C1, C2, and C3) to allow for multiplexing. In the current experiment, murine oocytes were dual-stained and quantified using a C2 Nanog probe and a C3 Pou5f1 probe. These probes were selected based on the reported expression of Nanog and Pou5f1 in oocytes and embryos. At the conclusion of the hybridization steps, oocytes were placed in drops of anti-fade mounting media for application to histological slides. Confocal images were used to quantify the number of punctate fluorescent signals which represent individual mRNAs. In addition to quantifying the mRNAs, imaging also showed the spatial distribution of the specific mRNA in the cell, which other RNA quantification methods are unable to achieve. This technique proved to have low variability within an experimental group allowing the use of smaller numbers of oocytes in each experimental group to identify significant differences between experimental groups3.

<table><tbody><tr><td>(&amp;plusmn;)-&amp;alpha;-Lipoic acid</td><td>Sigma-Aldrich</td><td>T1395</td><td>Alpha Lipoic Acid</td></tr><tr><td>Albumin, Bovine Serum, Low Fatty Acid</td><td>MP Biomedicals, LLC</td><td>199899</td><td>FAF BSA</td></tr><tr><td>BD 10mL TB Syringe</td><td>Becton, Dickinson and Company</td><td>309659</td><td>10 mL syringe</td></tr><tr><td>BD PrecisionGlide Needle</td><td>Becton, Dickinson and Company</td><td>305109</td><td>27 1/2 gauge needle</td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma-Aldrich</td><td>C7902</td><td>CaCl2-2H2O</td></tr><tr><td>Citric acid</td><td>Sigma-Aldrich</td><td>C2404</td><td>Citrate</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G6152</td><td>Glucose</td></tr><tr><td>Disodium phosphate</td><td></td><td></td><td>Na2HPO4</td></tr><tr><td>Easy Grip Petri Dish</td><td>Falcon Corning</td><td>351008</td><td>35 mm dish</td></tr><tr><td>Edetate Disodium</td><td>Avantor</td><td>8994-01</td><td>EDTA</td></tr><tr><td>Extra Fine Bonn Scissors</td><td>Fine Science Tools</td><td>14084-08</td><td>Straight, Sharp/Sharp, non-serrated, 13mm cutting edge scissors</td></tr><tr><td>Fetal Bovine Serum</td><td>Atlanta biologicals</td><td>S10250</td><td>FBS</td></tr><tr><td>Gentamicin Reagent Solution</td><td>gibco</td><td>15710-064</td><td>Gentamicin</td></tr><tr><td>GlutaMAX-I (100X)</td><td>gibco</td><td>35050-061</td><td>Glutamax</td></tr><tr><td>Gold Seal Micro Slides</td><td>Gold Seal</td><td>3039</td><td>25 x 75mm slides</td></tr><tr><td>Gonadotropin, From Pregnant Mares&#039; Serum</td><td>Sigma</td><td>G4877</td><td>eCG</td></tr><tr><td>hCG recombinant</td><td>NHPP</td><td>AFP8456A</td><td>hCG</td></tr><tr><td>Hyaluronidase, Type IV-S: From Bovine Testes</td><td>Sigma-Aldrich</td><td>H3884</td><td>Hyaluronidase</td></tr><tr><td>Jewelers Style Forceps</td><td>Integra</td><td>17-305X</td><td>Forceps 4-3/8&quot;, Style 5F, Straight, Micro Fine Jaw</td></tr><tr><td>L-(+)-Lactic Acid, free acid&amp;nbsp;</td><td>MP Biomedicals, LLC</td><td>190228</td><td>L-Lactate</td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Sigma-Aldrich</td><td>M2773</td><td>MgSO4-7H2O</td></tr><tr><td>MEM Nonessential Amino Acids</td><td>Corning&amp;nbsp;</td><td>25-025-Cl</td><td>NEAA</td></tr><tr><td>Microscope Cover Glass</td><td>Fisher Scientific</td><td>12-542-C</td><td>25 x 25x 0.15 mm cover slips</td></tr><tr><td>Mm-Nanog-O2-C2 RNAscope Probe</td><td>Advanced Cell Diagnostics</td><td>501891-C2</td><td>Nanog Probe</td></tr><tr><td>Mm-Pou5f1-O1-C3 RNAscope Probe</td><td>Advanced Cell Diagnostics</td><td>501611-C3</td><td>Pou5f1 Probe</td></tr><tr><td>MOPS</td><td>Sigma-Aldrich</td><td>M3183</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>Paraformaldehyde</td></tr><tr><td>PES 0.22 um Membrane -sterile</td><td>Millex-GP</td><td>SLGP033RS</td><td>0.22 um filters</td></tr><tr><td>Polyvinylpyrrolidone</td><td>Sigma-Aldrich</td><td>P0930</td><td>PVP</td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>60128</td><td>KCl</td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma-Aldrich</td><td>60218</td><td>KH2PO4</td></tr><tr><td>Prolong Gold antifade reagent</td><td>invitrogen</td><td>P36934</td><td>Antifade reagent without DAPI</td></tr><tr><td>RNAscope DAPI</td><td>Advanced Cell Diagnostics</td><td>320858</td><td>DAPI</td></tr><tr><td>RNAscope FL AMP 1</td><td>Advanced Cell Diagnostics</td><td>320852</td><td>Amplifier 1</td></tr><tr><td>RNAscope FL AMP 2</td><td>Advanced Cell Diagnostics</td><td>320853</td><td>Amplifier 2</td></tr><tr><td>RNAscope FL AMP 3</td><td>Advanced Cell Diagnostics</td><td>320854</td><td>Amplifier 3</td></tr><tr><td>RNAscope FL AMP 4 ALT A</td><td>Advanced Cell Diagnostics</td><td>320855</td><td>Amplifier 4 ALT A</td></tr><tr><td>RNAscope FL AMP 4 ALT B</td><td>Advanced Cell Diagnostics</td><td>320856</td><td>Amplifier 4 ALT B</td></tr><tr><td>RNAscope FL AMP 4 ALT C</td><td>Advanced Cell Diagnostics</td><td>320857</td><td>Amplifier 4 ALT C</td></tr><tr><td>RNAscope Fluorescent Multiplex Detection Reagents Kit</td><td>Advanced Cell Diagnostics</td><td>320851</td><td>FISH Reagent Kit</td></tr><tr><td>RNAscope Probe 3-plex Negative Control Probe</td><td>Advanced Cell Diagnostics</td><td>320871</td><td>Negative Control</td></tr><tr><td>RNAscope Probe 3-plex Positive Control</td><td>Advanced Cell Diagnostics</td><td>320881</td><td>Positive Control</td></tr><tr><td>RNAscope Probe Diluent</td><td>Advanced Cell Diagnostics</td><td>300041</td><td>Probe Diluent</td></tr><tr><td>RNAscope Protease III</td><td>Advanced Cell Diagnositics</td><td>322337</td><td>Protease III</td></tr><tr><td>RNAscope Protease III &amp;amp; IV Reagent Kit</td><td>Advanced Cell Diagnostics</td><td>322340</td><td>FISH Protease Kit</td></tr><tr><td>RNAscope Protease IV</td><td>Advanced Cell Diagnostics</td><td>322336</td><td>Protease IV</td></tr><tr><td>S/S Needle with Luer Hub 30G</td><td>Component Supply Co.</td><td>NE-301PL-50</td><td>blunt 30 gauge needle</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S6297</td><td>NaHCO3</td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S6191</td><td>NaCl</td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>306576</td><td>NaOH</td></tr><tr><td>Sodium pyruvate, &gt;= 99%</td><td>Sigma-Aldrich</td><td>P5280</td><td>Pyruvate</td></tr><tr><td>Solution 6 Well Dish</td><td>Agtechinc</td><td>D18</td><td>6 well dish</td></tr><tr><td>Taurine</td><td>Sigma-Aldrich</td><td>T8691</td><td>Taurine</td></tr><tr><td>Tissue Culture Dish</td><td>Falcon Corning</td><td>353002</td><td>60 mm dish</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td>Triton X-100</td></tr></tbody></table>

use of single molecule fluorescent in situ hybridization (sm-fish) to quantify and localize mrnas in murine oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking&#160;computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash&#160;buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Upon the completion of the protocol, the result will be individual images from confocal z-series (Figure 4A and Figure 5), stitched images (Figure 4C), and mRNA counts (Figure 4B). When multiplexing is performed, there will also be merged images showing the label for two different mRNAs (Fig...

Watch this Scientific Journal Video about Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes at JoVE.com

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), ...

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Research

JoVE Journal

Genetics

10.6K Views.  University of Nebraska-Lincoln. To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.

Video: Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

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

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

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

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

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;

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 ...

Biochemistry

Detection and Quantification of Single mRNA Molecules in Drosophila

Studying Muscle Transcriptional Dynamics at Single-molecule Scales in Drosophila

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

Author Spotlight: Investigating mRNA Spatial Distribution in Drosophila Muscle Tissue 

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

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

Developmental Biology

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

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

Biology

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...

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

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

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

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

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Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

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

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Studying Muscle Transcriptional Dynamics at Single-molecule Scales in Drosophila

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

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