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

我们感谢 Daniel R. Larson 博士在安装和使用现场查找和跟踪计划13方面提供的慷慨帮助, 并感谢内布拉斯加州林肯大学显微镜核心公司为共聚焦显微镜成像提供的技术支持。这项研究是内布拉斯加州林肯市内布拉斯加州林肯大学农业研究司的一项贡献, 并得到 UNL 舱口基金的支持 (Neb-26-206 加入号-232435 和 Neb-26-23加入号-1013511)。

内布拉斯加州林肯大学动物护理和使用机构委员会审查并批准了动物程序, 所有方法都是按照相关准则和条例进行的。在这项研究中, CD-1 外繁殖的小鼠有正常的啮齿类动物食物和水;它们保持在12:12 的黑暗周期: 光循环中。
1. 准备所需的介质
<ol><li>对于基介质 (OMM), 加入 100 mM 氯化钠、5 mM KCl、0.5 mM kh 2 po 4 和 1.7Mm cl-2H 2o 至 100 ml无菌水. 注: OM...

<ol>
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该协议中的一系列小步骤将确保 Mrna 的成功荧光和准确计数。首先, 在收集和固定卵母细胞后, 必须立即执行该协议。请注意, PVP 被添加到4% 的甲醛固定缓冲液中, 以防止卵母细胞相互粘附。我们发现, 在卵母细胞的收集和固定后, 有必要立即进行实验。任何延迟都会导致更低的荧光信号, 从而导致笔录的低估。这在一定程度上是由于 RNA 降解。一次不应将超过20个卵母细胞转移到6井板中的一个井, 每...

逆转录酶聚合酶链反应 (PCR) 已成为 mRNA 定量的黄金标准。目前使用的是数字 PCR (dPCR)1和定量实时 Pcr( qpcr) 2 两种检测方法。在两种 PCR 技术中, dPCR 的灵敏度高于 qPCR, 表明它可用于测量单个细胞中的 mRNA 丰度。然而, 在我们手中, dPCR 分析低丰度 Mrna 池5至10个卵母细胞每个实验样本产生的数据具有低重现性和高变异3。这可能是由于与 RNA 提取和逆转录酶效率相关的实验错误。Rna 测序也使用单个小鼠和人类卵母细胞4,5进行。这种技术需要图书馆生成所需的 cDNA 扩增步骤, 这可能会增加实验组内的可变性。此外, 低丰度成绩单可能无法检测到。虽然测序价格在过去几年有所下降, 但由于生物信息学分析的成本很高, 测序价格仍然可能高得令人望而却步。最后, mRNA 定位是一个动态过程, 空间变化对蛋白质功能有影响。因此, 我们开始采用一种技术, 将产生准确和可重复的定量措施和定位单个卵母细胞中的单个 Mrna。
分支 dna 与荧光原位杂交结合, 可放大荧光信号, 而不是放大 nnacdna, 从而能够检测单个细胞7,8,9中的单个 mrna。该检测是通过一系列的杂交, 扩增 (使用分支 DNA), 和荧光标记步骤, 以放大荧光信号7。该技术从结合 18---------------------------------------------------------------------------------------------为每个转录记录设计了15到20对探针对, 以确保目标转录的特殊性。Mrna 特异性杂交之后是前放大器和放大器探头, 形成分支配置。大约400个标签荧光与每个放大器结合在一起, 从而使荧光增加 8000倍, 从而可以检测到单个 Mrna (图 1)11。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59414/59414fig1.jpg"> 图 1: sm-fish 协议的原理图.结果显示了转录特异性探针、分支 DNA 放大器和荧光体与靶向 mRNA 的顺序杂交。<a href="https://www.jove.com/files/ftp_upload/59414/59414fig1large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
以往的研究使用单分子荧光原位杂交 ( SM - FISH ) 局部β - 肌动蛋白 Mrna 在单个神经元 12 和人状瘤病毒 dna 在宫颈癌细胞系 7。计算机软件 "现场查找和跟踪程序" 可识别单个点点荧光信号, 并已成功地用于量化每个单元3、13 中的 mrna 数量。
根据神经元12中 mrna 检测的结果, 我们假设 sm-fish 将被证明是一个有用的工具, 用于定量小鼠卵母细胞和胚胎的转录水平, 包括低丰度 mrna。然而, 该技术是优化使用与粘附固定细胞和甲醛固定石蜡嵌入 (FFPE) 组织切片。卵母细胞不能粘附在滑块上, 即使它们被涂上多-l-赖氨酸。此外, 当在市售的试剂盒3中使用一些专有缓冲液时, 它们比体细胞和组织切片更脆弱, 从而导致细胞裂解。为了克服这些挑战, 卵母细胞被固定, 并在缓冲液滴之间手动转移。此外, 还更换了试剂盒中的渗透和洗涤缓冲液, 以减少细胞裂解。预先设计的探头与 FISH 套件一起购买, 或可以要求提供具体的成绩单。每个专有探针集都可以在三个荧光通道 (C1、C2 和 C3) 中的一个中使用, 以允许多路复用。在目前的实验中, 使用 C2 Nanog 探针和 C3 Pou5f1 探针对小鼠卵母细胞进行了双重染色和定量。这些探针是根据报道的纳诺格和Pou5f1在卵母细胞和胚胎中的表达情况选择的。在杂交步骤结束时, 卵母细胞被放置在防褪色安装介质的滴中, 用于组织学幻灯片。共聚焦图像被用来量化表示单个 Mrna 的点点荧光信号的数量。除了对 Mrna 进行量化外, 成像还显示了细胞中特定 mRNA 的空间分布, 而其他 RNA 定量方法是无法实现的。这项技术被证明在实验组中具有较低的变异性, 允许在每个实验组中使用较少数量的卵母细胞来识别实验组3之间的显著差异。

<table border="0" cellpadding="0" cellspacing="0" style="width:608px;" width="607">
	<tbody>
		<tr height="42">
			<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>
		</tr>
		<tr height="62">
			<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>
		</tr>
		<tr height="83">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Citric acid</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:123px;">C2404</td>
			<td>Citrate</td>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
		<tr height="62">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<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>
		<tr height="62">
			<td height="62" style="height:62px;width:223px;">RNAscope FL AMP 2</td>
			<td style="width:91px;">Advanced Cell Diagnostics</td>
			<td style="width:123px;">320853</td>
			<td>Amplifier 2</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:223px;">RNAscope FL AMP 3</td>
			<td style="width:91px;">Advanced Cell Diagnostics</td>
			<td style="width:123px;">320854</td>
			<td>Amplifier 3</td>
		</tr>
		<tr height="62">
			<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>
		</tr>
		<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>
		</tr>
		<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

目前用于量化卵母细胞和胚胎中 mRNA 的方法包括数字逆转录聚合酶链反应 (dPCR)、定量、实时 RT-PCR (RT-qPCR) 和 RNA 测序。当使用单个卵母细胞或胚胎进行这些技术时, 低拷贝 Mrna 无法可靠地检测到。为了克服这个问题, 卵母细胞或胚胎可以聚集在一起进行分析;然而, 这往往会导致样品之间的高变异性。在这个协议中, 我们描述了使用分支 DNA 化学的荧光原位杂交 (FISH) 的使用。此技术标识单个单元格中 Mrna 的空间模式。当该技术与现场查找和跟踪计算机软件相结合时, 细胞中 Mrna 的丰度也可以量化。使用这种技术, 实验组内的变异性降低, 检测实验组之间显著差异所需的卵母细胞和胚胎较少。商用分枝 dna SM-FISH 试剂盒已经过优化, 可检测切片组织或幻灯片上粘附细胞中的 Mrna。然而, 卵母细胞不能有效地粘附在滑块和一些试剂盒中的一些试剂过于苛刻, 导致卵母细胞裂解。为了防止这种裂解, 对 FISH 试剂盒进行了一些修改。具体而言, 为卵母细胞和胚胎的免疫荧光而设计的卵母细胞渗透和洗涤缓冲液取代了专有的缓冲液。在6孔板中进行了具有探头和放大器的渗透、洗涤和孵化, 并在协议末尾使用安装介质将卵母细胞放置在幻灯片上。这些修改能够克服商业上可获得的试剂盒的限制, 特别是卵母细胞裂解。为了准确、可重复地计算单个卵母细胞中 Mrna 的数量, 使用了计算机软件。该协议共同代表了 PCR 和测序的替代方案, 用于比较单个细胞中特定转录的表达。

协议完成后, 结果将是来自共聚焦 z 系列(图 4 a 和图 5)、拼接图像(图 4A)和 mrna 计数(图 4A) 的单个图像。执行多路复用时, 还将有显示两个不同 Mrna 标签的合并图像(图 5)。MRNA 计数是使用斐济生成的缝合图像<stro...

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

为了可重复地计数单个卵母细胞中的 Mrna 数量, 对非粘附细胞的单分子 RNA 荧光原位杂交 (RNA-FISH) 进行了优化。卵母细胞被收集, 与转录特异性探针杂交, 并使用图像定量软件进行量化。

单分子荧光原位杂交 (SM-FISH) 在小鼠卵母细胞中 Mrna 的定量和定位

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

这种技术不仅允许定位，而且允许在单个卵母细胞和胚胎中对候选mRNA进行定量。与其他检测（包括 PCR）相比，该技术的完成可产生低变异的可重复数据。演示这个程序的将是凯尔西·提姆，一个研究生在我的实验室。从编写所需的材料和五至八周大的雌性小鼠开始，如文本协议所述。使用 70% 乙醇清洁鼠标。暴露腹腔，想象女性生殖道。用钳子握住卵巢，从卵巢周围取出子宫韧带和多余的脂肪组织。切开子宫的卵巢。然后，将卵巢卵巢对放在35毫米的培养皿中，放在温暖的保持介质中。取出卵巢和周围的任何脂肪组织。用半英寸27针撕开卵巢的肿胀的安培。在撕裂和积积细胞卵母细胞复合物的位点推开卵巢将被驱逐。使用口移液器将排卵卵母细胞转移到含有含海龙酶的持有介质的100微升滴中。为了转移积液细胞，移液器的元相两个卵母细胞积液细胞复合物在含有持有介质的 hyaluronidase 中上下与口移液器。一旦它们没有积液细胞，使用口腔移液器将每个卵母细胞转移到含有仅持有介质的洗涤液滴中。对每个洗涤液滴重复此数。不要转移支离破碎或透明的卵母细胞。要进行 SM-FISH 染色，请先在包含 500 微升固定缓冲液的六井板的单个井中固定卵母细胞。将20个卵母细胞淹没在井中。在室温下在固定缓冲液中孵育卵母细胞20分钟。如文本协议所述，洗涤卵母细胞后，在室温下在渗透缓冲液中孵育卵母细胞30分钟。再次洗涤后，将卵母细胞转移到80微升的预稀释蛋白酶三缓冲液，在室温下30分钟。在探针稀释剂中稀释 Nanog、Pou5f1 和 DapB 的加热探测集 1 到 50。在80微升的转录器中孵育卵母细胞，特异性探针在40摄氏度下孵育两小时。当卵母细胞在探针和 AMP 缓冲器中时，它们往往会浮。至关重要的是，你通过轻轻地将卵母细胞推入缓冲液来淹没卵母细胞。将卵母细胞转移到500微升的洗涤缓冲液，并在室温下孵育10分钟。现在，在扩增缓冲液中按顺序孵育卵母细胞。首先，在80微升AMP1中孵育卵母细胞，在40摄氏度下孵育30分钟。然后在室温下将卵母细胞转移到500微升洗涤缓冲液10分钟。接下来，在80微升AMP2中孵育卵母细胞，在40摄氏度下孵育15分钟。洗完卵母细胞后，在AMP3的80微升中孵育卵母细胞，在40摄氏度下孵育30分钟，然后进行最后清洗。最后，在40摄氏度下将卵母细胞加入80微升的AMP4FL中，15分钟。将 12 微升防褪色安装介质移入幻灯片中心，无需向试剂添加气泡。将母细胞用尽可能少的洗涤缓冲液转移到安装介质中。最后，应用盖单。使用 Z 步对共体显微镜对三维卵母细胞进行图像成像，并保存文本协议中描述的图像。将 ND2 文件拖动到斐济并选择超堆栈。单击"图像"选项卡，选择"颜色"，然后单击"拆分通道"以分隔 ND2 文件的荧光通道。为每个荧光通道中卵母细胞的每个 Z 切片生成单独的 TIFF 文件。为此，请单击"图像"选项卡，选择"堆栈"，然后单击"堆叠到图像"。然后，单击"图像"选项卡，选择"类型"，然后单击 RGB 颜色将每个 Z 切片转换为单独的 RGB 颜色图像。将每个转换的图像另存为 TIFF 文件。将每个荧光通道的单个卵母细胞的图像放在新文件夹中，以避免在缝合过程中出现混淆。按文本协议中所述对文件进行规范化后，单击"插件"选项卡，选择"拼接"，然后单击网格集合。从下拉菜单中选择"顺序图像"，然后单击"确定"。浏览目录并选择包含单个卵母细胞在一个波长上的所有 Z 切片图像的文件夹。单击"确定"。将拼接图像底部的滑块移动到使用波长的适当颜色通道。通过单击"图像"、选择"类型"和"单击 RGB 颜色"，创建最终的 RGB 拼接图像。将拼接的图像转换为 32 位最大投影图片。为此，请单击"图像"，选择"类型"，然后单击 32 位。将此图像另存为新的 TIFF 文件。在现场查找和跟踪程序中打开 32 位拼接图像。选择"本地化"下拉列表并单击"本地化"，这将计算图像中发现的点数。此处显示的是使用现场查找和跟踪的 mRNA 的量化。蓝色箭头指向阈值以上的正信号。白色箭头显示阈值以下的荧光点，因此不计算。随后使用标准数据分析工具对mRNA计数进行了分析。中间 Z 系列图像的代表性图像显示为 Pouf51 和 Nanog。在元相两个主轴上对齐的染色体的DAPI染色以白色显示。该协议收集的数据显示，775个Pouf51转录和113个纳米转录本在元相两个卵母细胞。重要的是，在标有DapB探针的卵母细胞中未检测到任何斑点，这些斑点充当负对。当使用非粘附细胞时，重要的是从SM-FISH试剂盒中经验确定蛋白酶缓冲液的最佳稀释。mRNA检测使用1xPBS中未稀释和几种稀释蛋白酶缓冲液浓度的滴定曲线进行测试。本协议中使用的蛋白酶稀释为1至8，因为它显示了元相两个卵母细胞中Pouf51 mRNA平均荧光表达的最低变异。可执行靶蛋白的并发免疫荧光，以便将mRNA与RNA结合蛋白共同定位。mRNA与RNA结合蛋白的共同定位使研究人员能够确定调节卵母细胞或胚胎内mRNA储存、转化和降解的机制。

Research

JoVE Journal

Genetics

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

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

光遗传学随机突变用组蛋白miniSOG在<em&gt;℃。线虫</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

科研

遗传学

Detection of Copy Number Alterations Using Single Cell Sequencing

单细胞测序拷贝数改变的检测

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

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

通过多色荧光跨染色体畸变稳定的检测<em&gt;原位</em&gt;杂交（渔业部）和光谱核型分析（SKY）的照射小鼠

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

Generation of Fluorescent Protein Fusions in Candida Species

在荧光蛋白融合的一代<em&gt;念珠菌</em&gt;物种

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

激光局部补骨脂素加合物的单分子分析

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

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

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

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 ...

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

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

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

全挂载原位杂交法可视化蜱内脏微生物群

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 Analysis in Budding Yeast Vegetative Growth and Meiosis

单分子荧光原位杂交 (smFISH) 在发芽酵母营养生长和减数分裂中的分析

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

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

RNA原位杂交法定量分析小鼠脑切片中的替代前 mRNA 拼接方法

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 ...