我们要感谢Miyako Shigeta的出色的技术支持。这项工作得到了JSS KAKENHI（授权号25461252）的部分支持。

所有动物实验方案均由Jichi Medical University动物伦理委员会批准，并按照"Jichi Medical University Guide for Laboratory Animals"的实验动物使用和护理指南进行。 腹膜样本收集<ol><li>收集以下物品:50 ml离心管，棉花浸泡在异氟烷，软木塞，磷酸盐缓冲盐水（PBS），手术剪刀和镊子的培养皿中。 </li><li>用过量的异氟烷安乐死大鼠，然后用70％乙醇喷洒大鼠的腹部皮肤，并将大鼠放在软木塞背面。 </li><li>使用剪刀和镊子在腹部皮肤，肌肉和腹膜中进行纵向切口。 </li><li>使用镊子拾取腹膜，并在其上部沿着隔膜下方的最下肋骨上方进行水平切口，并在其上侧面使用手术剪刀。接下来，使用手术剪刀进行横向纵向切口以从身体移除腹膜。然后用PBS在培养皿中洗涤。 </li><li>切割并修剪20毫克（3-5毫米2 ）的腹膜样品，这是适合以下步骤的尺寸，使用手术剪刀和镊子。 </li></ol> 2.从腹膜样品中纯化总RNA 注意:在这里，使用玻璃均化器和生物聚合物切碎系统将微量20mg的腹膜膜样品在微量离心旋转柱中均质化。然后，使用基于二氧化硅膜的旋转柱分离来自腹膜样品的总RNA。 <ol><li>收集以下物品:在微量离心旋转塔10中的 1.5或2.0mL微量离心管，100％乙醇，氯仿，玻璃均化器，冰，生物聚合物切碎系统</s，基于二氧化硅膜的旋转塔10 ，苯酚/胍基裂解试剂，包括胍和乙醇的洗涤缓冲液（洗涤缓冲液1）和包含乙醇的洗涤缓冲液（洗涤缓冲液2）。 </li><li>将20毫升腹膜样品放入玻璃匀浆器中，加入700微升苯酚/胍基裂解液。 </li><li>通过将杵慢慢地压在样品上，使腹膜膜样品均匀化。在冰上重复此过程数十次，直到腹膜膜样品完全溶解于苯酚/胍基裂解试剂中。 </li><li>为了进一步均化，将匀浆的裂解物转移到2.0mL收集管中的微量离心旋转柱中的生物聚合物切碎系统。然后在4℃下以14,000xg离心3分钟。 </li><li>将匀浆的裂解液转移到新的微量离心管中。 </li><li>向匀浆的裂解液中加入140μL氯仿，并盖上浴缸安全地然后，通过倒置将管混合15秒。 </li><li>在室温下孵育样品2-3分钟。然后在4℃下以12,000 xg离心15分钟。 </li><li>将上清液（通常为300μL）转移到新的微量离心管中，不会干扰沉淀物，并将其体积（通常为450μL）的100％乙醇加入其中。然后，通过涡旋混合样品5秒。 </li><li>将最多700μL样品吸取到置于2.0 mL收集管中的基于硅胶的旋转柱中。关闭列的顶盖。然后，以15,000 xg离心15秒。离心后，放弃收集管中的流通。 </li><li>将700μL洗涤缓冲液1加入到基于二氧化硅膜的旋转柱中以严格洗涤样品。关闭列的顶盖。然后，以15,000 xg离心15秒。离心后，放弃收集管中的流通。 </li><li>将500μL洗涤缓冲液2加入到二氧化硅膜中基于rane的旋转柱以除去任何痕量的盐。关闭列的顶盖。然后，以15,000 xg离心15秒。离心后，放弃收集管中的流通。 </li><li>重复2.11。 </li><li>再次以15,000xg离心二氧化硅膜基旋转柱1分钟。离心后，放弃收集管中的流通。 </li><li>将二氧化硅膜旋转柱放入新的1.5 mL收集管中。然后，将25μL无RNase的水转入柱中。关闭列的顶盖。将样品在室温下放置5分钟。然后，以15,000 xg离心1分钟。 </li><li>将含有总RNA的25μL洗脱液转移到新的微量离心管中。然后将其存放在-80°C使用前。 </li></ol> 3.逆转录RNA 注意:参考和遵守定量实时PCR发布的最低限度信息（MIQE）指南鼓励更好的实践，并帮助获得可靠和明确的结果12 。 注意:这里，使用逆转录酶，poly（A）聚合酶和oligo-dT引物逆转录总共1.0μg的分离的RNA。 <ol><li>收集以下物品:1.5 mL微量离心管;八孔条管;无RNase的水;冰;逆转录酶试剂盒（见材料表） 11 。 </li><li>准备一个主混合物溶液，其中含有2.0μL的10x核酸混合物，2.0μL的逆转录酶混合物（来自试剂盒）和4.0μL5x高规格缓冲液，总共每管8.0μL。 </li><li>将8.0μL主混合液（从试剂盒）分配到每个管中。 </li><li>使用分光光度计测量总RNA的量。将从腹膜膜样品纯化的1.0μg分离的总RNA添加到每个管中。 注意:可以使用qRT-PCR进行将总RNA作为模板，无逆转录用于纯化的总RNA的质量控制。如果任何DNA被污染，则通过qRT-PCR观察到意想不到的扩增产物。 </li><li>每个管中加入不含RNase的水，总共20μL。然后通过移液彻底混合并离心15秒。 </li><li>在37℃下孵育样品60分钟。立即在95°C孵育5分钟。 注意:该步骤可以在热循环仪中进行。 </li><li>将cDNA转移到新的微量离心管中，用无RNase的水稀释10倍（1:10）。 </li><li>将稀释的cDNA暂时储存在冰上，长期在-80℃下使用。 </li></ol> miRNA的qRT-PCR 注:使用插层染料进行miRNA的qRT-PCR。在这里，RNA引物，U6小核2（RNU6-2），miRNA-142-3p，miRNA-21-5p，miRNA-221-3p，miRNA-223-3p，miRNA-327和miRNA-34a-5p被使用。 <ol><li>收集以下物品:1.5mL微量离心管，qRT-PCR的96孔反应板，96孔反应板的粘合膜，miRNA特异性引物，基于绿色染料的PCR试剂盒（参见材料表） 11和a实时PCR仪。 </li><li>准备一个主混合物，其中含有12.5μL的2x PCR主混合物，2.5μL的5μM每个miRNA引物溶解在不含核酸酶的水中，每个孔用的是1.25μL的10x通用引物。 </li><li>将22.5μL的主混合物分配到96孔板的每个孔中。 </li><li>向每个孔中加入2.5μL模板cDNA。 </li><li>用96孔反应板的粘合膜密封板。然后，以1,000×g离心30秒。 </li><li> 使用实时PCR仪器和软件运行PCR循环程序如下。 <ol><li>将板置于实时PCR仪中。在实时PCR软件中，定义实验性质（输入实验名称，选择"96孔"为实验型仪器，选择"2 - ΔΔCT方法"进行定量方法，选择"SYBR绿色试剂"作为试剂检测目标序列，选择"标准斜坡速度"为仪器运行）。 </li><li>然后，为每个孔中的样品和目标miRNA分配名称。样品总是经过一式两份测试，以获得足够的数据用于验证结果。选择参考样本和内源性对照。选择"无"用作被动参考的染料。 </li><li>在实时PCR软件中按照以下说明输入反应体积为"20μL"和以下PCR循环条件:在95°C预温育15 min，然后在94°C变性15 s的40个循环，在55℃退火30秒，在70℃延伸30秒。 </li></ol></li><li>分析qRT-PCR数据u唱实时PCR仪的软件。确认由软件自动确定的阈值和基线在每个孔中都是合适的。 </li><li>检查每个样品中目标miRNA的阈值循环。阈值循环是放大曲线和阈值线之间的交点。然后，将目标miRNA的表达水平与RNU6-2作为内源性对照标准化，并使用2 - ΔΔCT方法计算目标miRNA的相对表达水平。 注意:应注意内源性对照miRNA表达水平的稳定性。在该实验中，RNU6-2被证实在高水平表达并且在每个组中表达相对不变。 </li></ol>

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作者宣称他们没有利益冲突。

使用本手册中提出的方案，大鼠腹膜中的miRNA成功纯化并使用qRT-PCR进行检测。 qRT-PCR数据分析的可靠性取决于纯化的miRNA的质量。因此，可以在qRT-PCR前通过260nm处的吸光度与280nm处的吸光度比检查miRNA的纯度，这可以使用分光光度计测量。当使用qRT-PCR不能获得miRNA的显着扩增时，模板cDNA的浓度可能会增加。每个miRNA特异性引物的浓度也可以增加。 确定miRNA表达水平有几种替代方?...

微小RNA（miRNA）是转录后调节信使RNA（mRNA）表达的短的非编码RNA。 miRNAs表达的变化调节许多在各种病理状况（包括癌症，炎症，代谢紊乱和纤维化）2,3,4,5,6,7,8中发挥关键作用的mRNA的表达。因此，miRNA具有潜在的新生物标志物和治疗靶标2,3,4,5,6,7,8。已经在各种大鼠中测定了miRNA表达谱器官和组织，包括肝脏，心脏，肺和肾脏9 。然而，用于纯化和检测大鼠腹膜中的miRNA的标准方法尚未得到很好的建立。 该方案的总体目标是成功地纯化和检测大鼠腹膜中的miRNA。首先，使用玻璃均化器将腹膜样品匀浆，然后在微量离心旋转塔10中暴露于生物聚合物破碎系统。接下来，使用基于二氧化硅膜的旋转柱10从腹膜样品纯化包括miRNA的总RNA。然后，使用逆转录酶，poly（A）聚合酶和oligo-dT引物11从纯化的总RNA合成cDNA。最后，使用插层染料11通过qRT-PCR测定miRNA的表达。这个协议的理由是b在以前的研究中，通过简单的过程8,10,11，显示了组织中miRNA的显着纯化和检测。据报道，在微量离心旋转柱和基于二氧化硅膜的旋转柱中使用生物聚合物粉碎系统可以从组织10中纯化高质量的总RNA。已经报道了使用逆转录酶，poly（A）聚合酶和oligo-dT引物从纯化的总RNA合成cDNA的方法，以及通过使用插入染料通过qRT-PCR检测miRNA表达的方法已经报道显示出高精度和灵敏度11 。此外，这是一个简单的过程，节省时间并防止技术错误。因此，该方案在需要在大范围病理条件下对大鼠腹膜中的miRNA进行高度准确和灵敏的检测的研究中是有用的。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>QIA shredder</td>
			<td>Qiagen</td>
			<td>79654</td>
			<td>biopolymer-shredding system in a micro centrifuge spin-column</td>
		</tr>
		<tr>
			<td>miRNeasy Mini kit</td>
			<td>Qiagen</td>
			<td>217004</td>
			<td>silica-membrane based spin column</td>
		</tr>
		<tr>
			<td>QIAzol Lysis Reagent</td>
			<td>Qiagen</td>
			<td>79306</td>
			<td>phenol/guanidine-based lysis reagent</td>
		</tr>
		<tr>
			<td>Buffer RLT</td>
			<td>Qiagen</td>
			<td>79216</td>
			<td>wash buffer 1</td>
		</tr>
		<tr>
			<td>Buffer RWT</td>
			<td>Qiagen</td>
			<td>1067933</td>
			<td>wash buffer 2</td>
		</tr>
		<tr>
			<td>miScript II RT kit&#160;</td>
			<td>Qiagen</td>
			<td>218161</td>
			<td>includes 10&#215; Nucleics Mix containing deoxynucleotides, ribonucleotide triphosphates, and oligo-dT primers; miScript Reverse Transcriptase Mix containing poly(A) polymerase and reverse transcriptase and; miScript HiSpec buffer</td>
		</tr>
		<tr>
			<td>miScript SYBR Green PCR kit</td>
			<td>Qiagen</td>
			<td>218073</td>
			<td>includes QuantiTect SYBR Green PCR Master Mix and miScript Universal Primer</td>
		</tr>
		<tr>
			<td>RNU6-2 primer&#160;&#160;</td>
			<td>Qiagen</td>
			<td>MS00033740</td>
			<td>not disclosed</td>
		</tr>
		<tr>
			<td>miRNA-142-3p primer</td>
			<td>Qiagen</td>
			<td>MS00031451</td>
			<td>5&#39;-UGUAGUGUUUCC 
			UACUUUAUGGA-3&#39;&#160;&#160;</td>
		</tr>
		<tr>
			<td>miRNA-21-5p primer</td>
			<td>Qiagen</td>
			<td>MS00009079</td>
			<td>5&#39;-UAGCUUAUCAG 
			ACUGAUGUUGA-3&#39;</td>
		</tr>
		<tr>
			<td>miRNA-221-3p primer</td>
			<td>Qiagen</td>
			<td>MS00003857</td>
			<td>5&#39;-AGCUACAUUGU 
			CUGCUGGGUUUC-3&#39;</td>
		</tr>
		<tr>
			<td>miRNA-223-3p primer</td>
			<td>Qiagen</td>
			<td>MS00033320</td>
			<td>5&#39;-UGUCAGUUUG 
			UCAAAUACCCC-3&#39;</td>
		</tr>
		<tr>
			<td>miRNA-34a-5p primer</td>
			<td>Qiagen</td>
			<td>MS00003318</td>
			<td>5&#39;-UGGCAGUGUCU 
			UAGCUGGUUGU-3&#39;</td>
		</tr>
		<tr>
			<td>miRNA-327 primer</td>
			<td>Qiagen</td>
			<td>MS00000805</td>
			<td>5&#39;-CCUUGAGGGG 
			CAUGAGGGU-3&#39;</td>
		</tr>
		<tr>
			<td>MicroAmp Optical 96 well reaction plate for qRT-PCR</td>
			<td>Thermo Fisher Scientific</td>
			<td>4316813</td>
			<td>96-well reaction plate</td>
		</tr>
		<tr>
			<td>MicroAmp Optical Adhesive Film&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>4311971</td>
			<td>adhesive film for 96-well reaction plate</td>
		</tr>
		<tr>
			<td>QuantStudio 12K Flex Flex Real-Time PCR system</td>
			<td>Thermo Fisher Scientific</td>
			<td>4472380</td>
			<td>real-time PCR instrument</td>
		</tr>
		<tr>
			<td>QuantStudio 12K Flex Software version 1.2.1.</td>
			<td>Thermo Fisher Scientific</td>
			<td>4472380</td>
			<td>real-time PCR instrument software</td>
		</tr>
		<tr>
			<td>methylglyoxal</td>
			<td>Sigma-Aldrich</td>
			<td>M0252</td>
			<td></td>
		</tr>
		<tr>
			<td>Midperic</td>
			<td>Terumo</td>
			<td>not assign&#160;</td>
			<td>peritoneal dialysis fluid</td>
		</tr>
		<tr>
			<td>Sprague&#8211;Dawley rats</td>
			<td>SLC</td>
			<td>not assign&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of microrna expression in peritoneal membrane of rats using quantitative real-time pcr

微小RNA（miRNA）是在转录后调节信使RNA表达的小型非编码RNA。已经在大鼠的各种器官和组织中研究了miRNA表达谱。然而，用于纯化miRNA的标准方法和其在大鼠腹膜中的表达检测尚未很好地建立。我们已经开发了一种有效和可靠的方法，使用定量实时逆转录聚合酶链反应（qRT-PCR）在大鼠腹膜中纯化和定量miRNA。该方案由四个步骤组成:1）净化腹膜样品; 2）从腹膜样品纯化包括miRNA的总RNA; 3）逆转录miRNA产生cDNA;和4）qRT-PCR检测miRNA表达。使用该方案，我们成功地确定了6种miRNA（miRNA-142-3p，miRNA-21-5p，miRNA-221-3p，miRNA-223-3p，miRNA-327和miRNA-34a-5p）的表达增加与对照组相比，大鼠腹膜纤维化模型的腹膜显着。该方案可用于研究许多病理条件下大鼠腹膜中miRNA表达谱。

这里提供的结果是基于我们以前报告的研究8 。我们调查了腹膜纤维化中的miRNA表达谱。腹膜纤维化是腹膜透析的主要并发症。其特征在于间皮细胞单层的损失和细胞外基质成分的过量积累，并且与腹膜破裂14,15相关。通过腹膜内注射100mL / kg腹膜透析液（2.5％葡萄糖，100mM NaCl，35mM乳酸钠，2mM CaCl 2和含有20mM甲基乙二醛的0.7mM MgCl <...

Watch this Scientific Journal Video about Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR at JoVE.com

Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR

在这里，我们提出了使用定量实时逆转录聚合酶链反应检测大鼠腹膜中microRNA表达的方案。该方法适用于在几种病理条件下研究大鼠腹膜中的microRNA表达谱。

使用定量实时PCR检测大鼠腹膜中的微小RNA表达

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been ...

detection-microrna-expression-peritoneal-membrane-rats-using

Research

JoVE Journal

Genetics

7.8K 次观看.  Saitama Medical Center, Jichi Medical University. 在这里，我们提出了使用定量实时逆转录聚合酶链反应检测大鼠腹膜中microRNA表达的方案。该方法适用于在几种病理条件下研究大鼠腹膜中的microRNA表达谱。 

视频: Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 nucleotides apart result in either the inclusion (S) or exclusion (&#916;S) of a single residue, Serine-701. Further downstream, splicing at a pair of alternative acceptor splice sites result in transcripts encoding either the 55 terminal residues of the transactivation domain (&#945;) or a truncated transactivation domain with 7 unique residues (&#946;). As outlined in this manuscript, measuring the proportions of STAT3's four spliced transcripts (S&#945;, S&#946;, &#916;S&#945; and &#916;S&#946;) was possible using absolute qPCR (quantitative polymerase chain reaction). The protocol therefore distinguishes and measures highly similar splice variants. Absolute qPCR makes use of calibrator plasmids and thus specificity of detection is not compromised for the sake of efficiency. The protocol necessitates primer validation and optimization of cycling parameters. A combination of absolute qPCR and efficiency-dependent relative qPCR of total STAT3 transcripts allowed a description of the fluctuations of STAT3 splice variants' levels in eosinophils treated with cytokines. The protocol also provided evidence of a co-splicing interdependence between the two STAT3 splicing events. The strategy based on a combination of the two qPCR techniques should be readily adaptable to investigation of co-splicing at other tandem splicing sites.

Tandem splicing events occur at sites less than 12 nucleotides apart. Quantifying ratios of such splice variants is feasible using an absolute quantitative PCR approach. This manuscript describes how splice variants of the gene STAT3, in which two splicing events results in Serine-701 inclusion/exclusion and &#945;/&#946; C-termini, can be quantified.

合并绝对和相对定量PCR数据来量化STAT3剪接变体成绩单

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 ...

科研

遗传学

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.

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 ...

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. In recent years, miRNA-mediated gene regulation has shown potential for treatment of neurological disorders caused by a toxic gain of function mechanism. However, efficient delivery to target tissues has limited its application. Here we used a transgenic mouse model for spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR), to test gene silencing by a newly identified AR-targeting miRNA, miR-298. We overexpressed miR-298 using a recombinant adeno-associated virus (rAAV) serotype 9 vector to facilitate transduction of non-dividing cells. A single tail-vein injection in SBMA mice induced sustained and widespread overexpression of miR-298 in skeletal muscle and motor neurons and resulted in amelioration of the neuromuscular phenotype in the mice.

Here we describe the delivery of microRNA using a recombinant adeno-associated virus serotype 9 in a mouse model of a neuromuscular disease. A single peripheral administration in mice resulted in sustained miRNA overexpression in muscle and motor neurons, providing an opportunity to study miRNA function and therapeutic potential in vivo.

重组腺相关病毒9型 MicroRNA 系统分娩治疗啮齿动物神经肌肉疾病

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. ...

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.

双向逆转录病毒整合位点PCR方法和定量数据分析工作流程

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase activity. Although TERT is named as a reverse transcriptase, structural and phylogenetic analyses of TERT demonstrate that TERT is a member of right-handed polymerases, and relates to viral RNA-dependent RNA polymerases (RdRPs) as well as viral reverse transcriptase. We firstly identified RdRP activity of human TERT that generates complementary RNA stand to a template non-coding RNA and contributes to RNA silencing in cancer cells. To analyze this non-canonical enzymatic activity, we developed RdRP assay with recombinant TERT in 2009, thereafter established in vitro RdRP assay for endogenous TERT. In this manuscript, we describe the latter method. Briefly, TERT immune complexes are isolated from cells, and incubated with template RNA and rNTPs including radioactive rNTP for RdRP reaction. To eliminate single-stranded RNA, reaction products are treated with RNase I, and the final products are analyzed with polyacrylamide gel electrophoresis. Radiolabeled RdRP products can be detected by autoradiography after overnight exposure.

Human telomerase reverse transcriptase (TERT) synthesizes not only telomeric DNA but also double-stranded RNA through RNA-dependent RNA polymerase activity. Here, we describe a newly established assay to detect RNA-dependent RNA polymerase activity of endogenous TERT.

人端粒酶逆转录蛋白 rna 依赖性 rna 聚合酶活性的半定量检测

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

CRISPR 的染色质环结构重组

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

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.

原位杂交法在小鼠心脏切片组织特异 miRNA 表达谱分析中的应用

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

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named &#8216;Worm-to-CT&#8217;, allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

In this article a high-throughput protocol for fast and reliable determination of gene expression levels in single or bulk C. elegans samples is described. This protocol does not require RNA isolation and produces cDNA directly from samples. It can be used together with high-throughput multiplexed nanofluidic real-time qPCR platforms.

使用纳米流体技术的单体和散装 C. 电子样品 中的高通量定量 RT-PCR

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly ...

Quantitative Real-Time PCR Evaluation of microRNA Expressions in Mouse Kidney with Unilateral Ureteral Obstruction

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to partially complementary target sites in the 3&#39; untranslated region (UTR) of messenger RNA (mRNA), which reduces the mRNA&#39;s translation and stability. The miRNA expression profiles in various organs and tissues of mice have been investigated, but standard methods for the purification and quantification of miRNA in mouse kidney have not been available. We have established an effective and reliable method for extracting and evaluating miRNA expression in mouse kidney with renal interstitial fibrosis by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The protocol required five steps: (1) creation of sham and unilateral ureteral obstruction (UUO) mice; (2) extraction of kidney samples from the UUO mice; (3) extraction of total RNA, which includes miRNA, from the kidney samples; (4) complementary DNA (cDNA) synthesis with reverse transcription from miRNA; and (5) qRT-PCR using the cDNA. Using this protocol, we successfully confirmed that compared to the controls, the expression of miRNA-3070-3p was significantly increased and those of miRNA-7218-5p and miRNA-7219-5p were significantly decreased in the kidneys of a mouse model of renal interstitial fibrosis. This protocol can be used to determine the miRNA expression in the kidneys of mice with UUO.

We describe a method for evaluating the microRNA expression in the kidneys of mice with unilateral ureteral obstruction (UUO) by quantitative reverse-transcription polymerase chain reaction. This protocol is suitable for studying kidney microRNA expression profiles in mice with UUO and in the context of other pathological conditions.

小鼠肾中微RNA表达与单体尿阻的定量实时PCR评价

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to ...

A Virtual Machine Platform for Non-Computer Professionals for Using Deep Learning to Classify Biological Sequences of Metagenomic Data

A variety of biological sequence classification tasks, such as species classification, gene function classification and viral host classification, are expected processes in many metagenomic data analyses. Since metagenomic data contain a large number of novel species and genes, high-performing classification algorithms are needed in many studies. Biologists often encounter challenges in finding suitable sequence classification and annotation tools for a specific task and are often not able to construct a corresponding algorithm on their own because of a lack of the necessary mathematical and computational knowledge. Deep learning techniques have recently become a popular topic and show strong advantages in many classification tasks. To date, many highly packaged deep learning packages, which make it possible for biologists to construct deep learning frameworks according to their own needs without in-depth knowledge of the algorithm details, have been developed. In this tutorial, we provide a guideline for constructing an easy-to-use deep learning framework for sequence classification without the need for sufficient mathematical knowledge or programming skills. All the code is optimized in a virtual machine so that users can directly run the code using their own data.

This tutorial describes a simple method to construct a deep learning algorithm for performing 2-class sequence classification of metagenomic data.

非计算机专业人员使用深度学习对元数据的生物序列进行分类的虚拟计算机平台

A variety of biological sequence classification tasks, such as species classification, gene function classification and viral host classification, are ...