Noninvasive collection of cell and region specific miRNA from heterogeneous tissues.

从异质组织中无创收集细胞和区域特异性 miRNA。

基本信息

批准号：
9047099
负责人：
Stanislav Karsten
金额：
$ 19.03万
依托单位：
NEUROINDX INC.
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-02-01 至 2018-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9047099
关键词：
Address Affect Animals Archives Area Attention Binding Biological Preservation Blood capillaries Brain Brain region Buffers Cell Culture Techniques Cell Separation Cells Code Collection Complementary DNA Complex Correlation Studies Data Development Disease Dissection Dissociation Ensure Equipment Evaluation Freezing Future Gel Gene Expression Genes Glass Histocompatibility Testing Human Immune Immunoassay Individual Label Lasers Legal patent Length Membrane Messenger RNA Methodology Methods MicroRNAs Microarray Analysis Microdissection Molecular Mus Neurosciences Nucleotides One-Step dentin bonding system Pathogenesis Pattern Performance Phase Physiologic pulse Plant Resins Plants Polysaccharides Procedures Process Proteins Protocols documentation RNA Regulation Regulator Genes Resolution Reverse Transcriptase Polymerase Chain Reaction Sampling Saponins Sepharose Solid Sorting - Cell Movement Source Specificity Specimen Surface System Technology Temperature Testing Time Tissue Banks Tissue Sample Tissues Training Translations Triton Tube Tweens Untranslated RNA Vacuum absorption base brain cell brain tissue capillary cell type cost cost efficient design follow-up gel electrophoresis human disease instrumentation non-Native novel strategies programs public health relevance research study sephadex technique development

项目摘要

DESCRIPTION (provided by applicant): Micro RNAs are crucial regulators of gene expression in plants and animals. Their complex expression patterns are associated with numerous human diseases, developmental programs and often appear to be cell and tissue specific. However, our understanding of miRNA expression and function in specific cell types and tissues is limited because of difficulty in obtaining appropriate cell and region specific specimen. Current methods of cell and region specific micro RNA isolation involve two major steps: first, tissue microdissection or dissociation with a follow up cell sorting and second, isolation of micro RNA molecules from acquired cells or tissue regions. This approach is time consuming and invasive. It results in the destruction of often valuable original tissue sample and demands for the use of costly equipment (e.g. laser based microdissection or flow sorting) and prior training. Here we will develop a noninvasive method for cell- and region- specific micro RNA collection from complex heterogeneous tissues. The approach is based on the use of polysaccharide resins and their specific regional acquisition. Prior pretreatment and even coating of the tissue sections with a resin permits micro RNA absorption directly from the tissue sample. Acquisition of cell- or region specific micro RNA is performed with our recently developed KuiqpicK technology via collection of the specific resin samples from the desired tissue regions or cells using carefully controlled vacuum pulses. Resin samples containing micro RNA are collected in the barrel of the disposable capillary unit (DCU) and transferred to a test tube where the captured miRNA is eluted and may be used for a variety of downstream applications, including large scale gene expression studies. The advantage of the proposed method over current approaches is manifold, including 1) direct one step acquisition of micro RNA from the specific cells, which eliminates the need for cell and tissue collection (i.e., microdissection); 2) preservation of the original tissue integrity, which permits its use for further experimentation such as immunohistology; 3) reduction in the time and cost; 4) possibility of immediate amplification and labeling of captured micro RNA; and 5) highly specific capturing of micro RNA species.

描述（由适用提供）：微RNA是植物和动物中基因表达的关键调节剂。它们的复杂表达模式与许多人类疾病，发育计划有关，并且通常是细胞和组织特异性的。但是，由于难以获得适当的细胞和特定区域标本，我们对特定细胞类型和组织中miRNA表达和功能的理解受到限制。当前的细胞和区域特异性微RNA分离的方法涉及两个主要步骤：首先，组织显微解剖或分离，并通过后续细胞分类，其次，从获得的细胞或组织区域分离微RNA分子。这种方法是耗时和侵入性的。它导致经常有价值的原始组织样本破坏，并要求使用昂贵的设备（例如，基于激光的显微解剖或流动分析）和事先培训。在这里，我们将开发一种从复杂异质组织中收集细胞和区域特异性微RNA的无创方法。该方法基于使用多糖树脂及其特定的区域采集。事先预处理甚至组织切片涂层树脂允许直接遭受组织样品的微RNA。通过使用精心控制的真空脉冲从所需的组织区域或细胞中收集特定的树脂样品，通过我们最近开发的Kuiqpick技术进行细胞或区域特异性微RNA的采集。在一次性毛细管（DCU）的桶中收集了含有微RNA的树脂样品，并转移到测试管中，其中捕获的miRNA被洗脱，可用于多种下游应用，包括大型基因表达研究。所提出的方法比当前方法的优点是多种方法，包括1）从特定细胞中直接从特定细胞中获得微RNA，这消除了对细胞和组织收集的需求（即微解答）； 2）保留原始组织完整性，该完整性允许其用于进一步实验，例如免疫组织学； 3）减少时间和成本； 4）立即放大和标记捕获的微RNA的可能性； 5）微RNA物种的高度特异性捕获。