?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.
Public Health Relevance Statement: Public Health Relevance: Micro RNAs (miRNAs) regulate expression of up to 80% of human protein coding genes. Moreover, recent data convincingly demonstrate that miRNAs are regulated in a complex cell- and tissue specific pattern. However our understanding of these complex cell-, tissue-, disease- specific and developmental regulation patterns is hampered by several technical limitations including difficulties of obtaining micro RNA from cell- and tissue- specific sources. Current methods involving tissue microdissection or dissociation with a follow up micro RNA isolation are cumbersome, expensive, time consuming and result in the destruction of an original tissue sample. Our approach proposed here permits noninvasive single step collection of cell- and tissue-specific micro RNA via the use of polysaccharide resins and a recently developed sample acquisition technology (KuiqpicK) utilizing controlled vacuum impulse. Briefly, the resin is evenly distributed over the surface of a target tissue section to absorb micro RNA. The subsequent area specific collection ensures acquisition of micro RNA from the desired tissue regions or cells. miRNA is easily extracted from the collected resin samples and may be used directly in a variety of downstream applications, including large scale gene expression studies. The approach is rapid and simple, and importantly permits preservation of potentially valuable tissue sample that may be used for additional immunoassays or protein studies. Phase I project will focus on the evaluation of various resins, optimization of micro RNA yields, quality assessment in comparison to the laser based microdissection techniques, and development of a streamlined protocol. Developed protocol will be integrated with KuiqpicK system. Further refinement and development of the commercial product will be performed in Phase II of this application.
Project Terms: absorption; Address; Affect; Animals; Archives; Area; Attention; base; Binding (Molecular Function); Biological Preservation; Blood capillaries; Brain; brain cell; Brain region; brain tissue; Buffers; capillary; Cell Culture Techniques; Cell Separation; cell type; Cells; Code; Collection; Complementary DNA; Complex; Correlation Studies; cost; Data; design; Development; Disease; Dissection; Dissociation; Ensure; Equipment; Evaluation; follow-up; Freezing; Future; Gel; gel electrophoresis; Gene Expression; Genes; Glass; Histocompatibility Testing; Human; human disease; Immune; Immunoassay; Individual; instrumentation; Label; Lasers; Legal patent; Length; Membrane; Messenger RNA; Methodology; Methods; Microarray Analysis; Microdissection; MicroRNAs; Molecular; Mus; Neurosciences; novel strategies; Nucleotides; One-Step dentin bonding system; Pathogenesis; Pattern; Performance; Phase; Physiologic pulse; Plant Resins; Plants; Polysaccharides; Procedures; Process; programs; Proteins; Protocols documentation; public health relevance; Regulation; Regulator Genes; research study; Resolution; Reverse Transcriptase Polymerase Chain Reaction; RNA; Sampling; Saponin; Saponins; sephadex; Sepharose; Solid; Solutions; Sorting - Cell Movement; Source; Specificity; Specimen; Surface; System; technique development; Technology; Temperature; Testing; Time; Tissue Banks; Tissue Sample; Tissues; Training; Translations; Triton; Tritons; Tube; Tweens; Untranslated RNA; Vacuum
