The effect of the mimic/inhibitor is determined by comparing this result with the gene expression in untransfected cells or cells transfected with a negative control. The expression of an endogenous gene, which is known to be a target of the miRNA under study, is measured after mimic/inhibitor transfection.The effect of the mimic/inhibitor is determined by comparing this to the result from cells transfected with the vector alone. After transfection, a reporter assay, such as a luciferase assay, is performed. miRNA mimic and/or inhibitor is cotransfected with the vector. A plasmid vector which carries a reporter gene such as luciferase and one or more miRNA binding sites in the 3' UTR is used as an miRNA target.Alternatively, the role of miRNAs in various pathways can be studied by examination of a specific phenotype following miRNA mimic or inhibitor transfection.ĭownstream analysis of the effect of miRNA mimic/inhibitor transfection is often performed using one of the following strategies: Reduced gene expression after transfection of an miRNA mimic or increased expression after transfection of an miRNA inhibitor provides evidence that the miRNA under study is involved in regulation of that gene. These experiments enable study of the biological effects of misregulation of individual miRNAs, as well as confirmation of specific genes as targets of individual miRNAs. Transfection of mimics, followed by downstream gene expression analysis or phenotypic analysis, is performed to elucidate the targets and roles of particular miRNAs. Mimics and inhibitors may also contain chemical modifications proposed to improve activity or in vivo stability. miRNA inhibitors are single-stranded (usually 21–25 nucleotides), modified RNAs which, after transfection, specifically inhibit miRNA function. MiRNA mimics are chemically synthesized, double-stranded RNAs (usually 18–24 nucleotides) which mimic mature endogenous miRNAs after transfection into cells. In animal miRNA, partial complementarity has made positive identification of true binding sites difficult and imprecise. Naturally occurring miRNA-binding sites are typically found in the 3' untranslated regions (UTRs) of target mRNAs. In addition, miRNAs can mediate mRNA destruction by rapid deadenylation and/or decapping. Mature miRNAs contribute to the regulation of endogenous genes, primarily by translational repression. The miRNA system is an endogenous mechanism of regulation of gene expression. It is believed that humans have more than 2000 miRNAs, and they are estimated to regulate as over two-thirds of human genes. In contrast to RNAi, the miRNA pathway focuses on regulating the cell’s own genes. Additionally, miRNA precursors (pre-miRNAs) are not completely double-stranded, but rather form hairpin-like structures that contain double-stranded regions. Unlike the double-stranded RNA that triggers RNAi, miRNAs are encoded in the genome. The discovery that cell-free miRNAs are detectable in serum and plasma, and that their expression varies as a result of disease, presents great potential for cell-free miRNA expression signatures to be used as biomarkers in disease diagnosis and prevention.īoth miRNA and siRNA pathways involve double-stranded RNA, but the source of these RNAs differs. Overwhelming evidence indicates that dysregulation of miRNA expression is a cause or indicator of several disease processes, including many cancers. MiRNAs play an important role in many biological processes, including differentiation and development, cell signaling, and response to infection.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |