Name: use of single molecule fluorescent in situ hybridization (sm-fish) to quantify and localize mrnas in murine oocytes
Uploaded: 2019-04-24T14:00:00
Description: 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

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....

<ol>
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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.

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			<td height="42" style="height:42px;width:223px;">(&#177;)-&#945;-Lipoic acid</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:123px;">T1395</td>
			<td style="width:172px;">Alpha Lipoic Acid</td>
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			<td height="62" style="height:62px;width:223px;">Albumin, Bovine Serum, Low Fatty Acid</td>
			<td style="width:91px;">MP Biomedicals, LLC</td>
			<td style="width:123px;">199899</td>
			<td>FAF BSA</td>
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			<td height="83" style="height:83px;width:223px;">BD 10mL TB Syringe</td>
			<td style="width:91px;">Becton, Dickinson and Company</td>
			<td style="width:123px;">309659</td>
			<td>10 mL syringe</td>
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		<tr height="83">
			<td height="83" style="height:83px;width:223px;">BD PrecisionGlide Needle</td>
			<td style="width:91px;">Becton, Dickinson and Company</td>
			<td style="width:123px;">305109</td>
			<td>27 1/2 gauge needle</td>
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			<td height="42" style="height:42px;width:223px;">Calcium chloride dihydrate</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:123px;">C7902</td>
			<td>CaCl2-2H2O</td>
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			<td height="42" style="height:42px;width:223px;">Citric acid</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:123px;">C2404</td>
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			<td height="42" style="height:42px;width:223px;">D-(+)-Glucose</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:123px;">G6152</td>
			<td>Glucose</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Disodium phosphate</td>
			<td style="width:91px;"></td>
			<td style="width:123px;"></td>
			<td>Na2HPO4</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Easy Grip Petri Dish</td>
			<td style="width:91px;">Falcon Corning</td>
			<td style="width:123px;">351008</td>
			<td>35 mm dish</td>
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			<td height="21" style="height:21px;width:223px;">Edetate Disodium</td>
			<td style="width:91px;">Avantor</td>
			<td style="width:123px;">8994-01</td>
			<td>EDTA</td>
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			<td height="42" style="height:42px;width:223px;">Extra Fine Bonn Scissors</td>
			<td style="width:91px;">Fine Science Tools</td>
			<td style="width:123px;">14084-08</td>
			<td>Straight, Sharp/Sharp, non-serrated, 13mm cutting edge scissors</td>
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			<td height="42" style="height:42px;width:223px;">Fetal Bovine Serum</td>
			<td style="width:91px;">Atlanta biologicals</td>
			<td style="width:123px;">S10250</td>
			<td>FBS</td>
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			<td height="21" style="height:21px;width:223px;">Gentamicin Reagent Solution</td>
			<td style="width:91px;">gibco</td>
			<td style="width:123px;">15710-064</td>
			<td>Gentamicin</td>
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			<td height="21" style="height:21px;width:223px;">GlutaMAX-I (100X)</td>
			<td style="width:91px;">gibco</td>
			<td style="width:123px;">35050-061</td>
			<td>Glutamax</td>
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			<td height="21" style="height:21px;width:223px;">Gold Seal Micro Slides</td>
			<td style="width:91px;">Gold Seal</td>
			<td style="width:123px;">3039</td>
			<td>25 x 75mm slides</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Gonadotropin, From Pregnant Mares&#39; Serum</td>
			<td style="width:91px;">Sigma</td>
			<td style="width:123px;">G4877</td>
			<td>eCG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">hCG recombinant</td>
			<td style="width:91px;">NHPP</td>
			<td style="width:123px;">AFP8456A</td>
			<td>hCG</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Hyaluronidase, Type IV-S: From Bovine Testes</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:123px;">H3884</td>
			<td>Hyaluronidase</td>
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			<td height="21" style="height:21px;width:223px;">Jewelers Style Forceps</td>
			<td style="width:91px;">Integra</td>
			<td style="width:123px;">17-305X</td>
			<td>Forceps 4-3/8&#34;, Style 5F, Straight, Micro Fine Jaw</td>
		</tr>
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			<td height="62" style="height:62px;width:223px;">L-(+)-Lactic Acid, free acid&#160;</td>
			<td style="width:91px;">MP Biomedicals, LLC</td>
			<td style="width:123px;">190228</td>
			<td>L-Lactate</td>
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			<td height="42" style="height:42px;width:223px;">Magnesium sulfate heptahydrate</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:123px;">M2773</td>
			<td>MgSO4-7H2O</td>
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			<td height="42" style="height:42px;width:223px;">MEM Nonessential Amino Acids</td>
			<td style="width:91px;">Corning&#160;</td>
			<td style="width:123px;">25-025-Cl</td>
			<td>NEAA</td>
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			<td height="42" style="height:42px;width:223px;">Microscope Cover Glass</td>
			<td style="width:91px;">Fisher Scientific</td>
			<td style="width:123px;">12-542-C</td>
			<td>25 x 25x 0.15 mm cover slips</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:223px;">Mm-Nanog-O2-C2 RNAscope Probe</td>
			<td style="width:91px;">Advanced Cell Diagnostics</td>
			<td style="width:123px;">501891-C2</td>
			<td>Nanog Probe</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:223px;">Mm-Pou5f1-O1-C3 RNAscope Probe</td>
			<td style="width:91px;">Advanced Cell Diagnostics</td>
			<td style="width:123px;">501611-C3</td>
			<td>Pou5f1 Probe</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">MOPS</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:123px;">M3183</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Paraformaldehyde</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:123px;">P6148</td>
			<td>Paraformaldehyde</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">PES 0.22 um Membrane -sterile</td>
			<td style="width:91px;">Millex-GP</td>
			<td style="width:123px;">SLGP033RS</td>
			<td>0.22 um filters</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Polyvinylpyrrolidone</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:123px;">P0930</td>
			<td>PVP</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Potassium chloride</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:123px;">60128</td>
			<td>KCl</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Potassium phosphate monobasic</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:123px;">60218</td>
			<td>KH2PO4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Prolong Gold antifade reagent</td>
			<td style="width:91px;">invitrogen</td>
			<td style="width:123px;">P36934</td>
			<td>Antifade reagent without DAPI</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:223px;">RNAscope DAPI</td>
			<td style="width:91px;">Advanced Cell Diagnostics</td>
			<td style="width:123px;">320858</td>
			<td>DAPI</td>
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		<tr height="62">
			<td height="62" style="height:62px;width:223px;">RNAscope FL AMP 1</td>
			<td style="width:91px;">Advanced Cell Diagnostics</td>
			<td style="width:123px;">320852</td>
			<td>Amplifier 1</td>
		</tr>
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			<td height="62" style="height:62px;width:223px;">RNAscope FL AMP 2</td>
			<td style="width:91px;">Advanced Cell Diagnostics</td>
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			<td>Amplifier 2</td>
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			<td>Amplifier 3</td>
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			<td height="62" style="height:62px;width:223px;">RNAscope FL AMP 4 ALT A</td>
			<td style="width:91px;">Advanced Cell Diagnostics</td>
			<td style="width:123px;">320855</td>
			<td>Amplifier 4 ALT A</td>
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		<tr height="62">
			<td height="62" style="height:62px;width:223px;">RNAscope FL AMP 4 ALT B</td>
			<td style="width:91px;">Advanced Cell Diagnostics</td>
			<td style="width:123px;">320856</td>
			<td>Amplifier 4 ALT B</td>
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		<tr height="62">
			<td height="62" style="height:62px;width:223px;">RNAscope FL AMP 4 ALT C</td>
			<td style="width:91px;">Advanced Cell Diagnostics</td>
			<td style="width:123px;">320857</td>
			<td>Amplifier 4 ALT C</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:223px;">RNAscope Fluorescent Multiplex Detection Reagents Kit</td>
			<td style="width:91px;">Advanced Cell Diagnostics</td>
			<td style="width:123px;">320851</td>
			<td>FISH Reagent Kit</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:223px;">RNAscope Probe 3-plex Negative Control Probe</td>
			<td style="width:91px;">Advanced Cell Diagnostics</td>
			<td style="width:123px;">320871</td>
			<td>Negative Control</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:223px;">RNAscope Probe 3-plex Positive Control</td>
			<td style="width:91px;">Advanced Cell Diagnostics</td>
			<td style="width:123px;">320881</td>
			<td>Positive Control</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:223px;">RNAscope Probe Diluent</td>
			<td style="width:91px;">Advanced Cell Diagnostics</td>
			<td style="width:123px;">300041</td>
			<td>Probe Diluent</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:223px;">RNAscope Protease III</td>
			<td style="width:91px;">Advanced Cell Diagnositics</td>
			<td style="width:123px;">322337</td>
			<td>Protease III</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:223px;">RNAscope Protease III &#38; IV Reagent Kit</td>
			<td style="width:91px;">Advanced Cell Diagnostics</td>
			<td style="width:123px;">322340</td>
			<td>FISH Protease Kit</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:223px;">RNAscope Protease IV</td>
			<td style="width:91px;">Advanced Cell Diagnostics</td>
			<td style="width:123px;">322336</td>
			<td>Protease IV</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">S/S Needle with Luer Hub 30G</td>
			<td style="width:91px;">Component Supply Co.</td>
			<td style="width:123px;">NE-301PL-50</td>
			<td>blunt 30 gauge needle</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Sodium bicarbonate</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:123px;">S6297</td>
			<td>NaHCO3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Sodium chloride</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:123px;">S6191</td>
			<td>NaCl</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Sodium hydroxide</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:123px;">306576</td>
			<td>NaOH</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Sodium pyruvate, &#62;= 99%</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:123px;">P5280</td>
			<td>Pyruvate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Solution 6 Well Dish</td>
			<td style="width:91px;">Agtechinc</td>
			<td style="width:123px;">D18</td>
			<td>6 well dish</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Taurine</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:123px;">T8691</td>
			<td>Taurine</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Tissue Culture Dish</td>
			<td style="width:91px;">Falcon Corning</td>
			<td style="width:123px;">353002</td>
			<td>60 mm dish</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Triton X-100</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:123px;">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), ...

This technique allows for not only localization, but also quantitation of candidate mRNAs in the individual oocytes and embryos. Compared with other assays, including PCR, completion of the technique results in reproducible data with low variation. Demonstrating the procedure will be Kelsey Timme, a graduate student in my laboratory.Begin this procedure with the preparation of the required material and female mice at five to eight weeks of age as described in the text protocol. 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. Then, place the ovary oviduct pair in warm holding medium in a 35 millimeter dish.Remove the ovary and any surrounding adipose tissue. Tear the swollen ampullae of the oviduct using a half inch 27 gauge needle. Push the oviduct at the site of the tear and cumulus cell oocyte complexes will be expelled.Using a mouth pipette to transfer the ovulated oocytes to the 100 microliter drop containing holding medium with hyaluronidase. To dislodge cumulus cells, pipette the metaphase two oocyte cumulus cell complexes up and down in the hyaluronidase containing holding medium with the mouth pipette. Once they are devoid of cumulus cells, use the mouth pipette to transfer each oocyte to a wash drop containing only holding medium.Repeat this for each wash droplet. Do not transfer fragmented or transparent oocytes. To perform SM-FISH staining, first fix oocytes in an individual well of a six well plate containing 500 microliters of fixation buffer.Submerge 20 oocytes or less in the well. Incubate the oocytes in fixation buffer for 20 minutes at room temperature. After washing the oocytes as described in the text protocol, incubate oocytes in permeablization buffer for 30 minutes at room temperature.Following another wash, transfer the oocytes to 80 microliters of pre-diluted protease three buffer for 30 minutes at room temperature. Dilute the warmed probed sets for Nanog, Pou5f1, and DapB one to 50 in probe diluent. Incubate oocytes in 80 microliters of the transcript specific probe for two hours at 40 degrees Celsius.When oocytes are in the probe and the AMP buffers, they tend to float. It is essential that you submerge the oocytes by gently pushing them into the buffer. Transfer the oocytes to 500 microliters of wash buffer and incubate for 10 minutes at room temperature.Now, incubate oocytes sequentially in amplification buffers. First, incubate oocytes in 80 microliters of AMP1 for 30 minutes at 40 degrees Celsius. Then transfer the oocytes to 500 microliters of wash buffer for 10 minutes at room temperature.Next, incubate oocytes in 80 microliters of AMP2 for 15 minutes at 40 degrees Celsius. After washing the oocytes as before, incubate the oocytes in 80 microliters of AMP3 for 30 minutes at 40 degrees Celsius followed by a final wash. Finally, add oocytes to 80 microliters of AMP4FL for 15 minutes at 40 degrees Celsius.Pipette 12 microliters of anti-fade mounting medium onto the center of a slide without adding bubbles to the reagent. Transfer oocytes with as little wash buffer as possible into the mounting medium. Finally, apply a cover slip.Image the three dimensional oocytes using Z-step confocal microscopy and save the images as described in the text protocol. Drag ND2 files into Fiji and choose Hyperstack. Click the Image tab, select Color, and click Split Channels to separate the fluorescent channels of the ND2 file.Generate individual TIFF files for each Z slice of the oocytes in each fluorescent channel. To do so, click the Image tab, select Stacks, and click Stack to Images. Then, click the Image tab, select Type, and click RGB Color to convert each Z slice to an individual RGB color image.Save each converted image as a TIFF file. Place images from a single oocyte for each fluorescent channel in a new folder to avoid confusion during stitching. After normalizing the files as described in the text protocol, click the Plugins tab, select Stitching, and click on Grid Collection.Select Sequential Images from the dropdown menu and click Okay. Browse the directory and select the folder containing all of the Z slice images for an individual oocyte at one wavelength. Click Okay.Move the slider at the bottom of the stitched image to the appropriate color channel for the wavelength used. Create the final RGB stitched image by clicking Image, selecting Type, and clicking RGB Color. Convert the stitched image to a 32-bit maximum projected picture.To do so, click Image, select Type, and click 32-bit. Save this image as a new TIFF file. Open the 32-bit stitched image in a spot finding and tracking program.Select the Localize dropdown and click Localize, which will calculate the number of spots found in the image. Shown here is the quantification of mRNAs using the spot finder and tracking. The blue arrow points to a positive signal above threshold.The white arrow shows a fluorescent spot below the threshold and therefore not counted. The mRNA counts were subsequently analyzed using a standard data analysis tool. Representative images of the middle Z series image are shown for Pouf51 and Nanog.DAPI staining of chromosomes aligned on the metaphase two spindle is shown in white. The data collected in this protocol showed 775 Pouf51 transcripts and 113 Nanog transcripts in metaphase two oocytes. Importantly, there were no spots detected in oocytes that are labeled with DapB probe, which served as the negative control.When using non-adherent cells, it is important to empirically identify the best dilution of protease buffer from the SM-FISH kit. mRNA detection was tested using a titration curve of undiluted and several diluted protease buffer concentrations in 1xPBS. Protease dilution used in this protocol was one to eight as it showed the lowest variation in the average fluorescent expression of Pouf51 mRNA in metaphase two oocytes.Concurrent immuno-fluorescence of a target protein can be performed in order to co-localize the mRNA with an RNA binding protein. Co-localization of the mRNAs with the RNA binding proteins allows investigators to determine mechanisms that regulate mRNA storage, translation, and degradation within an oocyte or embryo.

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Research

JoVE Journal

Genetics

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

Generation of Fluorescent Protein Fusions in Candida Species

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. Thus, molecular and genetic tools are needed to facilitate the study of their pathogenesis mechanisms. PCR-mediated gene modification is a straightforward and quick approach to generate epitope-tagged proteins to facilitate their detection. In particular, fluorescent protein (FP) fusions are powerful tools that allow visualization and quantitation of both yeast cells and proteins by fluorescence microscopy and immunoblotting, respectively. Plasmids containing FP encoding sequences, along with nutritional marker genes that facilitate the transformation of Candida species, have been generated for the purpose of FP construction and expression in Candida. Herein, we present a strategy for constructing a FP fusion in a Candida species. Plasmids containing the nourseothricin resistance transformation marker gene (NAT1) along with sequences for either green, yellow, or cherry FPs (GFP, YFP, mCherry) are used along with primers that include gene-specific sequences in a polymerase chain reaction (PCR) to generate a FP cassette. This gene-specific cassette has the ability to integrate into the 3'-end of the corresponding gene locus via homologous recombination. Successful in-frame fusion of the FP sequence into the gene locus of interest is verified genetically, followed by analysis of fusion protein expression by microscopy and/or immuno-detection methods. In addition, for the case of highly expressed proteins, successful fusions can be screened for primarily by fluorescence imaging techniques.

Generation of Fluorescent Protein Fusions in Candida Species

PCR-mediated gene modification can be used to generate fluorescent protein fusions in Candida species, which facilitates visualization and quantitation of yeast cells and proteins. Herein, we present a strategy for constructing a fluorescent protein fusion (Eno1-FP) in Candida parapsilosis.

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. ...

Single Molecule Analysis of Laser Localized Psoralen Adducts

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live cells. The DDR to helix distorting covalent DNA modifications, including interstrand DNA crosslinks (ICLs), is not as well defined. We have studied the DDR stimulated by ICLs, localized by laser photoactivation of immunotagged psoralens, in the nuclei of live cells. In order to address fundamental questions about adduct distribution and replication fork encounters, we combined laser localization with two other technologies. DNA fibers are often used to display the progress of replication forks by immunofluorescence of nucleoside analogues incorporated during short pulses. Immunoquantum dots have been widely employed for single molecule imaging. In the new approach, DNA fibers from cells carrying laser localized ICLs are spread onto microscope slides. The tagged ICLs are displayed with immunoquantum dots and the inter-lesion distances determined. Replication fork collisions with ICLs can be visualized and different encounter patterns identified and quantitated.

Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these diseases, do not exist in isolation when they colonize the vector; rather, they likely engage in interactions with resident microorganisms in the gut lumen. The vector microbiota has been demonstrated to play an important role in pathogen transmission for several vector-borne diseases. Whether resident bacteria in the gut of the Ixodes scapularis tick, the vector of several human pathogens including Borrelia burgdorferi, influence tick transmission of pathogens is not determined. We require methods for characterizing the composition of the bacteria associated with the tick gut to facilitate a better understanding of potential interspecies interactions in the tick gut. Using whole-mount in situ hybridization to visualize RNA transcripts associated with particular bacterial species allows for the collection of qualitative data regarding the abundance and distribution of the microbiota in intact tissue. This technique can be used to examine changes in the gut microbiota milieu over the course of tick feeding and can also be applied to analyze expression of tick genes. Staining of whole tick guts yield information about the gross spatial distribution of target RNA in the tissue without the need for three-dimensional reconstruction and is less affected by environmental contamination, which often confounds the sequencing-based methods frequently used to study complex microbial communities. Overall, this technique is a valuable tool that can be used to better understand vector-pathogen-microbiota interactions and their role in disease transmission.

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Here, we present a protocol to spatially and temporally assess the presence of viable microbiota in tick guts using a modified whole-mount in situ hybridization approach.

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these ...

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 ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To date, miRNAs have been implicated in a large number of biological and disease processes, which has signified the need for the reliable detection methods of miRNA transcripts. Here, we describe a detailed protocol for digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe-based miRNA detection, combined with protein immunostaining on mouse heart sections. First, we performed an in situ hybridization technique using the probe to identify miRNA-182 expression in heart sections from control and cardiac hypertrophy mice. Next, we performed immunostaining for cardiac Troponin T (cTnT) protein, on the same sections, to co-localize miRNA-182 with the cardiomyocyte cells. Using this protocol, we were able to detect miRNA-182 through an alkaline phosphatase based colorimetric assay, and cTnT through fluorescent staining. This protocol can be used to detect the expression of any miRNA of interest through DIG-labeled LNA probes, and relevant protein expression on mouse heart tissue sections.

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections.

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...