Figure 1
From: An integrated miRNA functional screening and target validation method for organ morphogenesis
Integrated miRNA functional screening and target validation method.
The method consists of two parts: (a) miRNA Functional Perturbation and (b) miRNA Target Validation (miR-PD). (a) The miRNA Functional Perturbation involves preparation of Nanoparticles by loading an amphiphilic peptide with miRNA antagomir (inhibitor) or mimic (activator) to functionally target a miRNA of interest. Alternatively, Liposome transfection may be used (see Methods). Murine embryonic organs of interest are micro-dissected and explanted into multi-well culture plates, either directly onto metal mesh in a modified Trowel-type system (e.g., intact manbibles, isolated mandibles, tongue) or directly onto floating Nuclepore filters (e.g., using salivary glands, kidneys, lungs). This system provides moderate analytical throughput by allowing use of different experimental conditions and phenotypic assays in parallel. (b) The miRNA Target Validation (miR-PD; miRNA-Pull Down Assay) involves preparations of organ lysates and incubation of the cytoplasmic fraction with biotinylated-mimic miRNA (mimic-biotin), followed by a streptavidin bead purification step to recover the biotinylated-miRNA mimic and its target mRNA transcripts. RISC: RNA Interference Silencing Complex (necessary for miRNA:mRNA interactions).
