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

 描述(申请人提供)：微RNA是植物和动物基因表达的重要调节因子。它们的复杂表达模式与许多人类疾病、发育程序有关，而且往往似乎是细胞和组织特有的。然而，由于难以获得合适的细胞和区域特异性标本，我们对miRNA在特定细胞类型和组织中的表达和功能的了解是有限的。目前提取细胞和区域特异性微小RNA的方法主要包括两个步骤：第一步是组织显微切割或分离，随后进行细胞分选；第二步是从获得的细胞或组织区域中分离出微小RNA分子。这种方法既耗时又具有侵入性。它导致破坏往往有价值的原始组织样本，并需要使用昂贵的设备(例如，基于激光的显微解剖或流动分选)和先前的培训。在这里，我们将开发一种非侵入性的方法，从复杂的异质组织中收集特定细胞和区域的微小RNA。这种方法是基于多糖树脂的使用及其特定的区域获取。组织切片的预先处理和均匀涂布 用树脂可以直接从组织样本中吸收微量RNA。使用我们最近开发的KuiqpicK技术，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本，可以获取特定于细胞或区域的microRNA。含有微小RNA的树脂样品被收集在一次性毛细管单元(DCU)的桶中，并转移到试管中，在试管中捕获的miRNA被洗脱，并可用于各种下游应用，包括大规模的基因表达研究。与现有方法相比，该方法的优点是多方面的，包括1)直接从特定细胞中一步获取微小RNA，这消除了对细胞和组织收集(即显微解剖)的需要；2)保留了原始组织的完整性，允许其用于进一步的实验，如免疫组织学；3)减少了时间和成本；4)立即扩增和标记捕获的微小RNA；以及5)高特异性捕获微小RNA物种。

 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.