Name: quantitative real-time pcr evaluation of microrna expressions in mouse kidney with unilateral ureteral obstruction
Uploaded: 2020-08-27T15:00:00
Description: Watch this Scientific Journal Video about Quantitative Real-Time PCR Evaluation of microRNA Expressions in Mouse Kidney with Unilateral Ureteral Obstruction at JoVE.com

我们感谢来自埃丹兹集团（https://en-author-services.edanzgroup.com/）的博士米歇尔·古德编辑了这份手稿草稿。

所有动物实验方案都经过吉池医科大学动物伦理委员会的批准，并按照《济池医科大学实验动物指南》中的实验动物使用和护理指南执行。
1. 假手术
<ol><li>准备以下物品：异氟兰、软木塞、脱毛霜、实验室湿巾、带磷酸盐缓冲盐水（PBS）、4-0尼龙、钳子、手术剪刀、棉签和8周大的C57BL/6雄性小鼠的培养皿。</li>
	<li>用1.5%等值的小鼠麻醉，并保持在1.5%。然后，在小鼠腹部涂抹...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+in+metabolism+and+metabolic+disorders.">MicroRNAs in metabolism and metabolic disorders.</a> Nature Review Molecular Cell Biology. 13 (4), 239-250 (2012).</li><li>Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRNA+in+Macrophage+Development+and+Function.">miRNA in Macrophage Development and Function.</a> Antioxidants and Redox Signaling. 25 (15), 795-804 (2016).</li><li>Sun, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+exosomal+miRNA+on+cancer+biology+and+clinical+applications.">Effect of exosomal miRNA on cancer biology and clinical applications.</a> Molecular Cancer. 17 (1), 147 (2018).</li><li>Zhou, W. C., Zhang, Q. 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W., Oris, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+commercially-available+automated+and+manual+extraction+kits+for+the+isolation+of+total+RNA+from+small+tissue+samples.">A comparison of commercially-available automated and manual extraction kits for the isolation of total RNA from small tissue samples.</a> BMC Biotechnology. 14, 94 (2014).</li><li>Mestdagh, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+quantitative+miRNA+expression+platforms+in+the+microRNA+quality+control+(miRQC)+study.">Evaluation of quantitative miRNA expression platforms in the microRNA quality control (miRQC) study.</a> Nature Methods. 11 (8), 809-815 (2014).</li><li>Kang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+real-time+PCR+assay+of+microRNAs+using+S-Poly(T),+a+specific+oligo(dT)+reverse+transcription+primer+with+excellent+sensitivity+and+specificity.">A novel real-time PCR assay of microRNAs using S-Poly(T), a specific oligo(dT) reverse transcription primer with excellent sensitivity and specificity.</a> PLoS One. 7 (11), 48536 (2012).</li><li>Lv, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+microRNAs+for+the+treatment+of+renal+fibrosis+and+CKD.">Therapeutic potential of microRNAs for the treatment of renal fibrosis and CKD.</a> Physiological Genomics. 50 (1), 20-34 (2018).</li><li>Liu, S. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MIQE+guidelines:+minimum+information+for+publication+of+quantitative+real-time+PCR+experiments.">The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.</a> Clinical Chemistry. 55 (4), 611-622 (2009).</li><li>Rao, X., Huang, X., Zhou, Z., Lin, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improvement+of+the+2^(-delta+delta+CT)+method+for+quantitative+real-time+polymerase+chain+reaction+data+analysis.">An improvement of the 2^(-delta delta CT) method for quantitative real-time polymerase chain reaction data analysis.</a> Biostatics Bioinformatics and Biomathematics. 3 (3), 71-85 (2013).</li><li>Chevalier, R. L., Forbes, M. S., Thornhill, B. 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上述 qRT-PCR 协议成功确定了目标 miRNA 的表达级别。在寻求获得有意义的 qRT-PCR 数据时，对提取的 miRNA 进行评估非常重要，为了在执行 qRT-PCR 之前确认 miRNA 的质量，应使用分光光度计检查 260 nm 的吸光率与 280 nm 的吸光率。如果 qRT-PCR 无法获得预期长度和熔化温度的单PCR放大或单模熔化曲线，则反应板的每个井中可能有DNA污染或底片二丁二苯。
miRNA的表达水平可以通过qRT-PCR以?...

MicroRNA （miRNA） - 导致信使RNA （mRNA）1降解和转录抑制的短，非编码RNA - 已被证明调节各种在生理学和疾病中具有关键作用的mRNA的表达（如炎症、纤维化、代谢紊乱和癌症）。因此，一些miRNA可能是各种疾病2，3，4，5,3的候选新型生物标志物和,治疗靶点。虽然小鼠器官和组织（包括脑、心、肺、肝、肾）的,miRNA表达特征,被描述为6、7、8、9、10，,7但还没有标准的方法对小鼠肾脏中患有肾间裂纤维化的miRNA进行提取和评价。9,108
我们设计了一个协议，以可靠地净化和检测肾间体纤维化小鼠肾脏中miRNA的表达。该协议涉及以下五个主要步骤。（1） 8周大的C57BL/6雄性小鼠分为接受假手术（对子）的小鼠和接受单体尿道阻塞（UUO）手术的小鼠，这些小鼠与肾间质纤维化有关。（2） 肾脏样本从假鼠和UOU小鼠中提取，在硅均质器中分别均质，然后转移到微离心柱11、12,上的生物聚合物粉碎系统。（3） 含有miRNA的总RNA通过硅膜基自旋柱12，13从肾脏样本中提取。（4） 利用这种提取的总RNA，使用逆转录酶、聚（A）聚合酶和寡聚-dT素，14，15，从总RNA中合成补充DNA（cDNA）。15（5） miRNA的表达通过定量逆转录聚合酶链反应（qRT-PCR）使用交算染料14，15,进行评估。
该协议是基于调查，取得了有意义的提取和评估在各种组织11，12，13，14，15，和协议中使用的生物聚合物粉碎系统被证明从组织在200611,12,13,14,15年12的高质量，总RNA。此外，先前的研究已经确认了协议各个方面（即具有逆转录酶、聚（A）聚合酶和从提取总RNA中提取的寡戈-dT质而合成的cDNA合成的准确性和敏感性），用于通过qRT-PCR测定具有中间染料14，15的miRNA表达。由于新协议具有简单、省时、减少技术误差等优点，因此该协议可用于需要准确、灵敏识别小鼠肾脏中miRNA配置文件的研究。此外，该议定书可适用于对许多病理状况的调查。
接下来，我们描述与UUO小鼠的miRNA表达特征的测定，它与肾脏间型纤维化有关。在人类中，肾间性纤维化是慢性肾病和末期肾病的常见和重要特征，无论其病因如何16，17。,17这种肾间质纤维化与肾衰竭的进展有关，它的特点是间质空间（如胶原蛋白、纤维素和α平滑肌蛋白）中细胞外基质成分的表达增加，17，,18。

<table>
	<tbody>
		<tr>
			<td>Qiagen</td>
			<td>79216</td>
			<td>Wash buffer 2</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen</td>
			<td>1067933</td>
			<td>Wash buffer 1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tokyo Laboratory Animals Science</td>
			<td>Not assigned</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Fisher Scientific</td>
			<td>4316813</td>
			<td>96-well reaction plate</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Fisher Scientific</td>
			<td>4311971</td>
			<td>Adhesive film for 96-well reaction plate</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen</td>
			<td>MS00001701</td>
			<td>5&#39;-UUAAUGCUAAUUGUGAUAGGGGU-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen</td>
			<td>MS00065141</td>
			<td>5&#39;-UUACACUCCAGUGGUGUCGGGU-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen</td>
			<td>MS00068067</td>
			<td>5&#39;-UGCAGGGUUUAGUGUAGAGGG-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen</td>
			<td>MS00068081</td>
			<td>5&#39;-UGUGUUAGAGCUCAGGGUUGAGA-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen</td>
			<td>217004</td>
			<td>Membrane anchored spin column in a 2.0 mL collection tube</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen</td>
			<td>218161</td>
			<td>Reverse transcriptase kit</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen</td>
			<td>218073</td>
			<td>Green dye-based PCR kit</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen</td>
			<td>79654</td>
			<td>Biopolymer spin columns in a 2.0 mL collection tube</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen</td>
			<td>79306</td>
			<td>Phenol/guanidine-based lysis reagent</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Fisher Scientific</td>
			<td>4472380</td>
			<td>Real-time PCR instrument</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Fisher Scientific</td>
			<td>4472380</td>
			<td>Real-time PCR instrument software</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen</td>
			<td>129112</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen</td>
			<td>MS00033740</td>
			<td>Not disclosed</td>
			<td></td>
		</tr>
		<tr>
			<td>Takara Bio</td>
			<td>9790B</td>
			<td>Silicon homogenizer</td>
			<td></td>
		</tr>
		<tr>
			<td>ASKUL</td>
			<td>GA04SW</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AS ONE</td>
			<td>ER1004NA45-KF2,62 -9968-32</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative real-time pcr evaluation of microrna expressions in mouse kidney with unilateral ureteral obstruction

MicroRNA （miRNA） 是单链、非编码的RNA分子，通常通过结合信使RNA（MRNA）的3'未翻译区域（UTR）中部分互补的目标位点来调节转录后水平的基因表达，从而降低mRNA的翻译和稳定性。对小鼠各种器官和组织中的miRNA表达特征进行了调查，但小鼠肾脏中miRNA的纯化和定量标准方法尚未提供。通过定量逆转录聚合酶链反应（qRT-PCR），为小鼠肾内质纤维化提取和评价miRNA表达建立了有效可靠的方法。议定书需要五个步骤：（1） 制造假和单边尿道阻塞（UUO）小鼠;（2） 制造假和单边尿道阻塞（UUO）小鼠;（3） 制造假和单边尿道阻塞（UUO）小鼠;（3） 制造假和单边尿道阻塞（UUO）小鼠;（3） 制造假尿（2） 从UOO小鼠中提取肾脏样本;（3） 从肾脏样本中提取总RNA，包括miRNA;（4） 与miRNA逆转录的互补DNA（cDNA）合成;和 （5） qrt - PCR 使用 cDNA。利用本协议，我们成功确认，与对联相比，miRNA-3070-3p的表达显著增加，米RNA-7218-5p和miRNA-7219-5p的肾间性纤维化小鼠模型的肾脏的表达显著减少。该协议可用于确定具有 UUO 的小鼠肾脏中的 miRNA 表达。

UUO小鼠模型是由左尿道结扎创建，如描述21 在8周大的雄性小鼠体重20-25克。用4-0丝线线进行双连结完全阻碍。镇痛（旋律5毫克/千克，皮下注射）在手术前和手术后2天每天施用。手术后8天，肾脏被收集，用PBS冲洗，解剖，并储存在液氮中作进一步分析。沙姆操作的老鼠充当对等对等对等对等对等。如果左肾有肝肾病，双结扎是成功的。基于使用此 UUO 模型获得的 miRNA qRT-PCR 数据?...

Watch this Scientific Journal Video about Quantitative Real-Time PCR Evaluation of microRNA Expressions in Mouse Kidney with Unilateral Ureteral Obstruction at JoVE.com

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

我们描述了一种通过定量逆转录聚合酶链反应评估具有单性尿道阻塞（UUO）小鼠肾脏微RNA表达的方法。该协议适用于研究小鼠的肾微RNA表达配置文件与UOU和其他病理条件的背景下。

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 ...

这种方法使得在患有多种病理条件的小鼠肾脏中提供微RNA表达成为可能，例如肾纤维化。该技术的主要优点是，它通过使用一个简单的过程，节省时间，防止技术错误，使微RNA表达分析具有高精度和灵敏度。要进行假手术，请使用手术剪刀和钳子在腹部的皮肤进行切口，将肌肉和腹膜从膀胱切到肋骨的左下边缘。用 PBS 润湿两个棉签，小心地将肠子拉到一边。放置湿润的拭子，以确定左肾和尿尿器。关闭膜膜，然后用4-0尼龙切口。要执行单侧尿道阻塞，或UOU，在腹部的皮肤切口，并削减肌肉和腹膜，证明为假手术。将 2.5 毫升注射器放在鼠标下方。用 PBS 润湿两个棉签，然后用钳子小心地将肠子拉到一侧，然后放置棉签以识别左侧尿尿器。用钳子抬起左肾。使用 4-0 丝绸将左尿尿器在两个相距约 1 厘米的地方进行开裂。切开两个粘结中心点的尿道，然后用4-0尼龙缝合线关闭膜和切口。切开膜后，如前所述，用钳子抬起，并使用手术剪刀在上边缘做侧切，然后沿着肋骨的最低边缘继续切口。识别左肾，用PBS回流，直到肾脏变成黄白。用手术剪刀切割左肾动脉和静脉，将其取出，并放在培养皿中，然后用PBS小心洗涤。用手术剪刀和钳子将肾脏切成大约10毫克的样品，将肾脏的每一块放入自己的1.5毫升微离心管中，并关闭管盖。在硅胶均质器中加入肾脏样品，并加入700微升苯基/瓜尼丁基解压试剂。慢慢地按压和扭转均质器对肾脏样本的纠缠，以均匀化它。重复压榨和扭曲，直到样品完全溶解在解解试剂中。为了进一步使样品均质化，将均质裂酸转移到生物聚合物旋转柱上，并在14，000次G下旋转三分钟，然后将沉淀的裂酸转移到未使用的1.5毫升微离心管中。将管中的利沙酸盐与140微升氯仿混合，并紧贴管盖。要混合酸盐和氯仿，反转管15次。在室温下孵育样品2至3分钟，然后在12，000次G下旋转，在4摄氏度下旋转15分钟。在不干扰沉淀物的情况下，将上流水液转移到新的1.5毫升微离心管中，并加入1.5倍的100%乙醇体积。漩涡混合物五秒钟。将样品的 700 微升加载到两毫升收集管中的膜锚定旋转柱上。关闭柱盖，在 15，000 次 G 下旋转柱，15 秒，然后将沉淀的裂酸扔掉收集管中沉淀的裂物。在两毫升收集管中，将 700 微升洗涤缓冲液添加到膜锚定旋转柱中，彻底清洗样品。然后重复离心并扔掉收集管。每次洗涤两次，用 500 微升的洗涤缓冲液重复洗涤。上次洗涤后，在两毫升收集管中旋转膜锚旋转柱，在 15，000 次 G 下旋转一分钟，然后扔掉沉淀的裂酸。将膜锚定旋转柱转移到新的 1.5 毫升管中。通过将 30 微升无 RNase 水添加到柱中，关闭柱盖，并在室温下保持 5 分钟，溶解总 RNA。然后在 15，000 次 G 下旋转列一分钟。将含有总RNA的样品转移到冰上新的微离心管中，通过分光光度法测量总RNA的浓度。根据手稿方向准备主混合溶液，并在八井管条的每个管中添加八微升。调整总RNA密度后，将总RNA的12微升等分放入每个管中，并关闭瓶盖。离心管15秒。将管子放入热循环器中，在37摄氏度下孵育60分钟。完成后，立即孵育5分钟，在95摄氏度下合成cDNA。将cDNA转移到新的1.5毫升微离心管中，用蒸馏水稀释10次。漩涡和离心管五秒钟，然后执行定量实时PCR，如文本手稿中所述。该协议用于在重达20至25克的8周大雄性小鼠中通过左尿道结扎创建单性尿道阻塞小鼠模型。用4-0丝线线进行双连结完全阻碍。肾脏在手术后八天内被收集。microRNA qRT-PCR数据表明，与对联小鼠肾脏相比，微RNA3070-3p水平显著提高，UUO小鼠肾脏的微RNA、7218-5p和微RNA7219-5p水平降低。尝试此协议时，请记住反复扭曲肾脏样本，直到它完全溶解在苯基/瓜尼丁基解压试剂中，以实现良好的RNA提取。

Research

JoVE Journal

Genetics

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

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

Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations

野生类型阻止PCR结合直接测序作为低频体细胞突变的检测的高灵敏度的方法

Accurate detection and identification of low frequency mutations can be problematic when assessing residual disease after therapy, screening for emerging ...

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

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 ...

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

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

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 ...

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 ...

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 ...

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

用于评估肿瘤细胞中的微RNA水平、功能和相关靶基因的体外协议

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

基于数字PCR的沙门氏菌健康高通量分析竞争指数

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

A Bioinformatics Pipeline to Accurately and Efficiently Analyze the MicroRNA Transcriptomes in Plants

生物信息学管道，用于准确、高效地分析植物中的微RNA转录

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in ...