Name: exosomal mirna analysis in non-small cell lung cancer (nsclc) patients' plasma through qpcr: a feasible liquid biopsy tool
Uploaded: 2016-05-27T15:00:00
Description: Watch this Scientific Journal Video about Exosomal miRNA Analysis in Non-small Cell Lung Cancer NSCLC Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool at JoVE.com

This study was also financed by MOCA (Multidisciplinair Oncologisch Centrum Antwerpen) grant 2014.

1.外来体隔离注意：从血浆样品外来体的分离，根据制造商的协议，并通过加入RNA酶处理与市售的试剂盒进行的，（所有这些步骤必须以个人和环境的保护设备及以下对生物样品的操纵特定的法律程序执行）。 <ol><li>取各1ml样品的血浆，保存于-80℃，并放置在冰上。 </li><li>离心样品以2,000×g下在室温下20分钟;这段话是为了去除细胞和碎片强制性的。 </li><li>传送包含清洁等离...

<ol>
	<li>Jemal, A., Siegel, R., Ward, E., Hao, Y., Xu, J., Thun, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics.">Cancer statistics.</a> Cancer J Clin. 59, 225-249 (2009).</li><li>Rolfo, C., 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=Novel+therapeutic+strategies+for+patients+with+NSCLC+that+do+not+respond+to+treatment+with+EGFR+inhibitors.">Novel therapeutic strategies for patients with NSCLC that do not respond to treatment with EGFR inhibitors.</a> Cancer Treat Rev. 40, 990-1004 (2014).</li><li>Rolfo, C., 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=Liquid+biopsies+in+lung+cancer:+the+new+ambrosia+of+researchers.">Liquid biopsies in lung cancer: the new ambrosia of researchers.</a> Biochim Biophys Acta. 1846, 539-546 (2014).</li><li>Thery, C., Zitvogel, L., Amigorena, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes:+composition,+biogenesis+and+function.">Exosomes: composition, biogenesis and function.</a> Nat Rev Immunol. 2, 569-579 (2002).</li><li>Fontana, S., Saieva, L., Taverna, S., Alessandro, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+proteomics+to+understanding+the+role+of+tumor-derived+exosomes+in+cancer+progression:+state+of+the+art+and+new+perspectives.">Contribution of proteomics to understanding the role of tumor-derived exosomes in cancer progression: state of the art and new perspectives.</a> Proteomics. 13 (10-11), 1581-1594 (2013).</li><li>Taverna, S., 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=Exosomal+shuttling+of+miR-126+in+endothelial+cells+modulates+adhesive+and+migratory+abilities+of+chronic+myelogenous+leukemia+cells.">Exosomal shuttling of miR-126 in endothelial cells modulates adhesive and migratory abilities of chronic myelogenous leukemia cells.</a> Mol Cancer. 11, (2014).</li><li>Taverna, S., 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=Curcumin+inhibits+in+vitro+and+in+vivo+chronic+myelogenous+leukemia+cells+growth:+a+possible+role+for+exosomal+disposal+of+miR-21.">Curcumin inhibits in vitro and in vivo chronic myelogenous leukemia cells growth: a possible role for exosomal disposal of miR-21.</a> Oncotarget. , (2015).</li><li>Garofalo, M., 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=MicroRNA+signatures+of+TRAIL+resistance+in+human+non-small+cell+lung+cancer.">MicroRNA signatures of TRAIL resistance in human non-small cell lung cancer.</a> Oncogene. 27, 3845-3855 (2008).</li><li>Rabinowits, G., Ger&#231;el-Taylor, C., Day, J. M., Taylor, D. D., Kloecker, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomal+microRNA:+a+diagnostic+marker+for+lung+cancer.">Exosomal microRNA: a diagnostic marker for lung cancer.</a> Clin Lung Cancer. 10 (1), 42-46 (2009).</li><li>Hu, J., 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=Human+miR-1228+as+a+stable+endogenous+control+for+the+quantification+of+circulating+microRNAs+in+cancer+patients.">Human miR-1228 as a stable endogenous control for the quantification of circulating microRNAs in cancer patients.</a> Int J Cancer. 135 (5), 1187-1194 (2014).</li><li>Alessandro, R., 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=Effects+of+carboxyamidotriazole+on+in+vitro+models+of+imatinib-resistant+chronic+myeloid+leukemia.">Effects of carboxyamidotriazole on in vitro models of imatinib-resistant chronic myeloid leukemia.</a> J Cell Physiol. 215, 111-121 (2008).</li><li>Zhao, Y., 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=Exosomes+Derived+from+Human+Umbilical+Cord+Mesenchymal+Stem+Cells+Relieve+Acute+Myocardial+Ischemic+Injury.">Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells Relieve Acute Myocardial Ischemic Injury.</a> Stem Cells Int. 2015, (2015).</li><li>Yoko, T., 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=Up-regulation+of+MDR'1and+induction+of+doxorubicin+resistance+by+histone+deacetylase+inhibitor+depsipeptide+(FK228)+and+ATRA+in+acute+promyelocytic+leukemia+cells.">Up-regulation of MDR'1and induction of doxorubicin resistance by histone deacetylase inhibitor depsipeptide (FK228) and ATRA in acute promyelocytic leukemia cells.</a> Blood. 107 (4), 1546-1554 (2006).</li></ol>

用该组合协议是可能的从非小细胞肺癌患者血浆执行外来体miRNA的分析。这些数据可反映疾病​​的状态，但是这需要通过进一步研究加以证实。 在这篇文章中所使用的协议是基于切体隔离商业套件。其原因是，其它已知的分离方法（ 例如 ，密度梯度超速离心）需要更多的人工程序，从而导致有限的标准化。然而，该协议的局限性之一是，与用于外来体隔离密度梯度?...

非小细胞肺癌（非小细胞肺癌）患者生存率低的主要原因是在先进的疾病和不良的早期检测1治疗的疗效有限。在EGFR基因活化突变，已在非小细胞肺癌的患者的亚群被发现是已知赋予灵敏度酪氨酸激酶抑制剂（TKI中）。不幸的是，大多数EGFR突变的非小细胞肺癌患者的发展TKI阻力位2。特别是在这种情况下，正确的诊断是必需的。在一般情况下，由于肿瘤的定位，在非小细胞肺癌获得活检组织并不总是可行的。因此，一些研究是使用非侵入性的方法来获得这些生物数据正在进行。外来体被描述为可能，以获得与非侵入性技术3的诊断和预后值进行调查液体活检组件。 外来体是纳米囊泡 - 电子商务（40 100纳米直径）ndocytic原点由既在生理和病理条件4种不同类型的细胞释放。它们可以携带蛋白质，脂质，mRNA和微小RNA。此外，一些研究表明，肿瘤衍生的外来体，其可影响肿瘤细胞，血管生成，基质细胞和胞外基质重构和耐药5的生长和存活的多效性作用。 微RNA（miRNA）是所涉及的转录后调控，结合靶mRNA的3&#39;-UTR以及导致mRNA的降解或到非翻译6短的非编码RNA。 miRNA的选择性排序成外来体已经描述。在这种情况下，最近证实， 在体外和体内中，miR-21（公知的miRNA用致癌效应）的姜黄素治疗7后由慢性髓性白血病细胞系释放外来的选择性包装。 该外来体miRNA谱是类似的原发肿瘤的miRNA谱和这个特征可在早期诊断和预后3被利用。具体地说，是表征，通过实时PCR一种可能性，与疾病的分子轮廓，相关选择的miRNA 例如 ，除其他8 EGFR突变。 这里，描述了非小细胞肺癌相关的miRNA 8-9的选定面板的分析，认为是从非小细胞肺癌患者的血液中释放的外来体分离。获得来自患者的知情同意报告后，12个NSCLC患者和6名健康对照的血浆，进行了分析。外来体分离按照生产商的方案用市售的试剂盒进行。外来体分离之前，血浆样品用RNA酶处理，以降低污染物循环的miRNA。我们决定来执行该分析用市售试剂盒，由于其他TECHNI有限标准化疑问句（ 例如 ，超离心）的临床样品时这是特别重要的。这种试剂盒包含一个蛋白酶K处理，这将降低RNA酶，并因此降低外来体裂解后降解外来体的miRNA的风险。被敏感，特异的实时PCR分析进行分析的microRNA;的mir-1228-3p用作内源的控制和数据进行标准化根据公式2 - ΔΔCT。控制值被用作基准和结果在对数标度示出。

<table border="0" cellpadding="0" cellspacing="0" width="884">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Total exosome isolation kit (from plasma)</td>
			<td style="width:159px;">&#160;Invitrogen</td>
			<td style="width:159px;">4484450</td>
			<td style="width:265px;">Use Proteinase K treatment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ribonuclease A</td>
			<td>Sigma-Aldrich</td>
			<td>R4875-100MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ALIX Antibody (3A9)</td>
			<td>Cell Signaling</td>
			<td>2171S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TSG-101 Antibody (C-2)</td>
			<td>SantaCruz Biotecnology</td>
			<td>SC-7964&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Mouse HRP 1ml&#160;&#160;</td>
			<td>Cell Signaling</td>
			<td>7076P2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNAspin Illustra mini kit 50</td>
			<td>GE HealthCare</td>
			<td>25-0500-71</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Taqman MicroRNA Reverse Transcription Kit</td>
			<td>Life Technologies</td>
			<td>4366596</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hsa-miR-1228-3p TaqMan MicroRNA assay</td>
			<td>Life Technologies</td>
			<td>002919</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hsa-miR-30b TaqMan MicroRNA assay</td>
			<td>Life Technologies</td>
			<td>000602</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hsa-miR-30c TaqMan MicroRNA assay</td>
			<td>Life Technologies</td>
			<td>000419</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hsa-miR-122 TaqMan MicroRNA assay</td>
			<td>Life Technologies</td>
			<td>002245</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hsa-miR-195 TaqMan MicroRNA assay</td>
			<td>Life Technologies</td>
			<td>000494</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hsa-miR-203 TaqMan MicroRNA assay</td>
			<td>Life Technologies</td>
			<td>000507</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hsa-miR-103 TaqMan MicroRNA assay</td>
			<td>Life Technologies</td>
			<td>000439</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hsa-miR-221 TaqMan MicroRNA assay</td>
			<td>Life Technologies</td>
			<td>000524</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hsa-miR-222 TaqMan MicroRNA assay</td>
			<td>Life Technologies</td>
			<td>002276</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Universal Master Mix II, no UNG</td>
			<td>Life Technologies</td>
			<td>4440043</td>
			<td></td>
		</tr>
	</tbody>
</table>

exosomal mirna analysis in non-small cell lung cancer (nsclc) patients' plasma through qpcr: a feasible liquid biopsy tool

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective tyrosine kinase inhibitors (TKIs). To optimize the use of these TKIs, the discovery of new biomarkers for early detection and disease progression is mandatory. These plasma-isolated exosomes can be used as a non-invasive and repeatable way for the detection and follow-up of these biomarkers. One ml of plasma from 12 NSCLC patients, with different mutations and treatments (and 6 healthy donors as controls), were used as exosome sources. After RNAse treatment, in order to degrade circulating miRNAs, the exosomes were isolated with a commercial kit and resuspended in specific buffers for further analysis. The exosomes were characterized by western blotting for ALIX and TSG101 and by transmission electron microscopy (TEM) analysis, the standard techniques to obtain biochemical and dimensional data of these nanovesicles. Total RNA extraction was performed with a high yield commercial kit. Due to the limited miRNA-content in exosomes, we decided to perform retro-transcription PCR using an individual assay for each selected miRNA. A panel of miRNAs (30b, 30c, 103, 122, 195, 203, 221, 222), all correlated with NSCLC disease, were analyzed taking advantage of the remarkable sensitivity and specificity of Real-Time PCR analysis; mir-1228-3p was used as endogenous control and data were processed according to the formula 2-&#916;&#916;ct 13. Control values were used as baseline and results are shown in logarithmic scale.

共有NSCLC患者12血浆和6名健康对照的与从血浆中分离的外来体商业试剂盒进行了处理。每个样品用蛋白质印迹分析，以评估所述外来体标记ALIX（96 kDa的）和TSG101（43 kDa）的处理。这些结果的一个例子示出了用于在图1 3的样品，这表明从血浆样品外来体隔离与此协议是可行的。 为了表征生物物理的样品外体，进行​...

Watch this Scientific Journal Video about Exosomal miRNA Analysis in Non-small Cell Lung Cancer NSCLC Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool at JoVE.com

Exosomal miRNA Analysis in Non-small Cell Lung Cancer NSCLC Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool

This protocol describes the feasibility to perform miRNA profiling in exosomes, released in plasma of NSCLC patients, through a commercial exosome isolation kit with Proteinase K and RNAse treatments, in order to avoid circulating miRNAs contamination and evaluate their biomarker features in NSCLC.

外来体miRNA的分析非小细胞肺癌（NSCLC）患者的血浆通过定量PCR：一种可行的液体活检工具

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective ...

Research

JoVE Journal

Medicine

Exosomal miRNA Analysis in Non-small Cell Lung Cancer (NSCLC) Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

科研

医学

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

这些模型在体外间皮间隙检测卵巢癌转移的早期步骤

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

A Simple Method of Mouse Lung Intubation

一个简单的方法小鼠肺插管

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

心肌缺血再灌注损伤的通过左前降支结扎小鼠模型

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

在前列腺癌的临床组织中miRNA的表达分析

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, ...

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

利用miRNA的miRNA表达的临床病理分析乳腺癌组织<em&gt;原位</em&gt;杂交

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

磷31磁共振波谱分析：一种测量工具<em&gt;在体内</em&gt;线粒体氧化磷酸化能力人骨骼肌

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

A Detailed Protocol for Physiological Parameters Acquisition and Analysis in Neurosurgical Critical Patients

神经外科危重患者生理参数采集与分析的详细方案

Intracranial pressure (ICP) monitoring is now widely used in neurosurgical critical patients. Besides mean ICP value, the ICP derived parameters such as ...