Name: detection of microrna expression in peritoneal membrane of rats using quantitative real-time pcr
Uploaded: 2017-06-27T14:00:00.000+00:00
Duration: 8 min 56 s
Description: Watch this Scientific Journal Video about Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR at JoVE.com

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><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&amp;nbsp;</td><td>Qiagen</td><td>218161</td><td>includes 10&amp;times; 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&amp;nbsp;&amp;nbsp;</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&#039;-UGUAGUGUUUCC&lt;br/&gt; UACUUUAUGGA-3&#039;&amp;nbsp;&amp;nbsp;</td></tr><tr><td>miRNA-21-5p primer</td><td>Qiagen</td><td>MS00009079</td><td>5&#039;-UAGCUUAUCAG&lt;br/&gt; ACUGAUGUUGA-3&#039;</td></tr><tr><td>miRNA-221-3p primer</td><td>Qiagen</td><td>MS00003857</td><td>5&#039;-AGCUACAUUGU&lt;br/&gt; CUGCUGGGUUUC-3&#039;</td></tr><tr><td>miRNA-223-3p primer</td><td>Qiagen</td><td>MS00033320</td><td>5&#039;-UGUCAGUUUG&lt;br/&gt; UCAAAUACCCC-3&#039;</td></tr><tr><td>miRNA-34a-5p primer</td><td>Qiagen</td><td>MS00003318</td><td>5&#039;-UGGCAGUGUCU&lt;br/&gt; UAGCUGGUUGU-3&#039;</td></tr><tr><td>miRNA-327 primer</td><td>Qiagen</td><td>MS00000805</td><td>5&#039;-CCUUGAGGGG&lt;br/&gt; CAUGAGGGU-3&#039;</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&amp;nbsp;</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&amp;nbsp;</td><td>peritoneal dialysis fluid</td></tr><tr><td>Sprague&amp;ndash;Dawley rats</td><td>SLC</td><td>not assign&amp;nbsp;</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

Universidad San Sebastian

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

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

科研

生物工程

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

实时定量PCR（qPCR的microRNA检测在前列腺肿瘤）

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

医学

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

小胶质细胞的小分子RNA检测在正常中枢神经系统的实时PCR技术在神经发炎

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

免疫与感染

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 ...

遗传学

Quantitative Real-Time Polymerase Chain Reaction Evaluation of MicroRNA Expression in Kidney and Serum of Mice with Age-Dependent Renal Impairment

MicroRNAs (miRNAs) are small, noncoding RNAs consisting of 21-25 bases. They are not translated into proteins but rather work to impede the functioning of their target messenger RNAs (mRNAs) by destabilizing them and disrupting their translation. Although the miRNA expression profiles in various mouse organs and tissues have been investigated, there have been no standard methods for purifying and quantifying mouse kidney and serum miRNAs. We have established an effective and reliable method for extracting and evaluating the miRNA expression in the serum and kidney of mice with age-dependent renal impairment. 
The method uses quantitative reverse-transcription-polymerase chain reaction (qRT-PCR), and the protocol requires six steps: (1) preparing senescence-accelerated mouse resistance 1 (SAMR1) mice and senescence-accelerated mouse prone (SAMP1) mice; (2) extracting serum samples from these mice; (3) extracting a kidney sample from each mouse; (4) extracting total RNA (including miRNA) from kidney and serum samples from each mouse; (5) the synthesis of complementary DNA (cDNA) with reverse transcription from the miRNA; (6) conducting a qRT-PCR using the cDNA obtained. 
This protocol was used to confirm that, compared to the controls, the expression of miRNA-7218-5p and miRNA-7219-5p was significantly changed in the kidney and serum of a mouse model of age-dependent renal impairment. This protocol also clarified the relationship between the kidney and serum of the mouse model of age-dependent renal impairment. This protocol can be used to determine miRNA expression in the kidney and serum of mice with age-dependent renal impairment.

We present a method for evaluating microRNA expression in the kidney and serum of mice with age-dependent renal impairment by quantitative reverse-transcription polymerase chain reaction.

年龄依赖性肾损伤小鼠肾脏和血清中MicroRNA表达的定量实时聚合酶链反应评估

MicroRNAs (miRNAs) are small, noncoding RNAs consisting of 21-25 bases. They are not translated into proteins but rather work to impede the functioning of ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

脑脊液的microRNA分析采用实时定量PCR技术

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

小鼠腹腔巨噬细胞的分离，以贯彻基因表达分析后Toll样受体刺激

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice

Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA, leading to the end-stage renal failure. In recent years, the involvement of microRNAs (miRNAs) has been reported in the pathogenesis of IgA nephropathy. However, there is no established method for profiling miRNAs in IgA nephropathy using small animal models. Therefore, we developed a reliable method for analyzing miRNA in the kidney of an IgA mouse model (HIGA mouse). The goal of this protocol is to detect the altered expression levels of miRNAs in the kidneys of HIGA mice when compared with the levels in kidneys of control mice. In brief, this method consists of four steps: 1) obtaining kidney samples from HIGA mice; 2) purifying total RNA from kidney samples; 3) synthesizing complementary DNA from total RNA; and 4) quantitative reverse transcription polymerase chain reaction (qRT-PCR) of miRNAs. Using this method, we successfully detected the expression levels of several miRNAs (miR-155-5p, miR-146a-5p, and miR-21-5p) in the kidneys of HIGA mice. This new method can be applied to other studies profiling miRNAs in IgA nephropathy.

microRNAs are involved in the pathogenesis of IgA nephropathy. We have developed a reliable method for detecting microRNA expression levels in the kidneys of an IgA nephropathy mouse model (HIGA mice). This new method will facilitate to check for miRNAs involvement in IgA nephropathy.

检测免疫球蛋白A肾病小鼠肾脏中的microRNA表达

Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA, leading to the end-stage renal ...

A Quantitative Detection Method for MicroRNAs in the Kidney of an Ischemic Kidney Injury Mouse Model

MicroRNAs (miRNAs) are involved in various disease states and are effective biomarkers for the early diagnosis of diseases and treatment in mice. However, standard protocols for the purification of miRNAs and detection of their expression in the kidneys of acute kidney injury (AKI) mice have not been well established. This study developed an effective and simple protocol to purify and quantify miRNAs in the kidneys of an AKI mouse model induced by renal ischemia-reperfusion using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). This protocol comprises five steps: 1) induction of AKI by renal ischemia-reperfusion, 2) harvesting of kidneys, 3) purification of total RNA, including miRNAs, from kidneys, 4) cDNA synthesis by reverse transcription of miRNA, and 5) qRT-PCR to detect miRNA expression. Using this protocol, the renal ischemia-reperfusion injury model can be generated with mild to severe forms of AKI. Additionally, if the procedure is followed properly, a consistent AKI model with minimal individual differences can be obtained. This qRT-PCR assay shows a very wide dynamic range and enables the discrimination of mature miRNAs, which can be accurately quantified with high specificity. This protocol can be used to study the miRNA expression profile in AKI kidneys.

This article presents a protocol for detecting microRNA expression in the kidneys of an acute kidney injury mouse model using quantitative real-time reverse-transcription polymerase chain reaction. This protocol emphasizes an ischemic kidney injury mouse model and the careful extraction of microRNA samples.

MicroRNAs (miRNAs) are involved in various disease states and are effective biomarkers for the early diagnosis of diseases and treatment in mice. However, ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

基于探针实时荧光定量PCR途径的小分子RNA定量检测

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

使用定量实时PCR检测大鼠腹膜中的微小RNA表达

摘要

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此视频中的章节

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidigm公司动态ArrayTM国际金融中心

实时定量PCR（qPCR的microRNA检测在前列腺肿瘤）

小胶质细胞的小分子RNA检测在正常中枢神经系统的实时PCR技术在神经发炎

小鼠肾中微RNA表达与单体尿阻的定量实时PCR评价

年龄依赖性肾损伤小鼠肾脏和血清中MicroRNA表达的定量实时聚合酶链反应评估

脑脊液的microRNA分析采用实时定量PCR技术

小鼠腹腔巨噬细胞的分离，以贯彻基因表达分析后Toll样受体刺激

检测免疫球蛋白A肾病小鼠肾脏中的microRNA表达

A Quantitative Detection Method for MicroRNAs in the Kidney of an Ischemic Kidney Injury Mouse Model

基于探针实时荧光定量PCR途径的小分子RNA定量检测

使用定量实时PCR检测大鼠腹膜中的微小RNA表达

摘要

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高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

实时定量PCR（qPCR的microRNA检测在前列腺肿瘤）

小胶质细胞的小分子RNA检测在正常中枢神经系统的实时PCR技术在神经发炎

小鼠肾中微RNA表达与单体尿阻的定量实时PCR评价

年龄依赖性肾损伤小鼠肾脏和血清中MicroRNA表达的定量实时聚合酶链反应评估

脑脊液的microRNA分析采用实时定量PCR技术

小鼠腹腔巨噬细胞的分离，以贯彻基因表达分析后Toll样受体刺激

检测免疫球蛋白A肾病小鼠肾脏中的microRNA表达

A Quantitative Detection Method for MicroRNAs in the Kidney of an Ischemic Kidney Injury Mouse Model

基于探针实时荧光定量PCR途径的小分子RNA定量检测

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidigm公司动态ArrayTM国际金融中心