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

 描述（由申请人提供）：微小RNA是植物和动物中基因表达的重要调节因子。它们复杂的表达模式与许多人类疾病、发育程序相关，并且通常表现为细胞和组织特异性。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理解是有限的。目前的细胞和区域特异性微小RNA分离方法包括两个主要步骤：第一，组织显微切割或解离，随后进行细胞分选;第二，从获得的细胞或组织区域分离微小RNA分子。这种方法是耗时和侵入性的。它导致通常有价值的原始组织样本的破坏，并需要使用昂贵的设备（例如基于激光的显微切割或流式分选）和预先培训。在这里，我们将开发一种非侵入性的方法，用于从复杂的异质组织中收集细胞和区域特异性的微小RNA。该方法基于多糖树脂的使用及其特定区域的获取。组织切片的预处理和均匀涂覆 用树脂可以直接从组织样本中吸收微小RNA。细胞或区域特异性微RNA的获取是通过我们最近开发的KuiqpicK技术进行的，通过使用仔细控制的真空脉冲从所需组织区域或细胞收集特异性树脂样品。将含有微小RNA的树脂样品收集在一次性毛细管单元（DCU）的筒中，并转移到试管中，在试管中洗脱捕获的miRNA，并可用于各种下游应用，包括大规模基因表达研究。所提出的方法相对于当前方法的优点是多方面的，包括1）从特定细胞直接一步获取微小RNA，这消除了对细胞和组织收集的需要（即，显微解剖）; 2）保持原始组织的完整性，这允许其用于进一步的实验，如免疫组织学; 3）减少时间和成本; 4）立即扩增和标记捕获的微小RNA的可能性;和5）高度特异性捕获微小RNA种类。

 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.