RNA interference (RNAi) technology is a powerful tool to study gene function in mammalian cells. The mechanism of RNAi is sequence-specific degradation of host mRNA by delivering double-stranded RNA (dsRNA) identical to the target into the cytoplasm. The degradation of target gene expression is achieved by an enzymatic pathway involving the endogenous RNA-induced silencing complex (RISC). Argonaute (Ago) proteins and small interfering RNAs (siRNAs) are the essential components of RISC. siRNA is processed from long dsRNA and guides the RISC to perfectly complementary target site in a mRNA molecule, where the endonucleolytically active Ago protein cleaves the mRNA. The cleaved mRNA is further degraded by other endogenous nucleases. The disadvantages of siRNA silencing include the short life span of siRNAs, the high cost of siRNAs, and the difficulty in efficiently delivering siRNAs to (non-dividing) primary cells. To address these limitations, vectors have been developed to generate short hairpin RNAs (shRNAs) that are processed intracellular into short dsRNAs with siRNA-like properties. shRNA is a single-strand molecule with stem-loop hairpin structure, which consists of a 19-29 base-pair region of dsRNA (the stem), a region of single-strand RNA (the loop), and a dinucleotide overhang at the 3′ end. The shRNA sequences are usually encoded in DNA vectors and can be introduced into cells by plasmid transfection or viral transduction. shRNA can mediate sustained gene silencing after stable integration of the vector into the host cell genome. In addition, the core silencing "hairpin" cassette can be easily inserted into retroviral, lentiviral or adenovirus vectors to facilitate shRNA delivery to various cell types.
High-throughput screening based on RNAi technology has become an efficient tool for functional genetic studies, especially for human cells where application of classical genetic tools is limited. RNAi screens can be applied to identify critical pathways of diseases, drug targets, and the therapeutic synergies. shRNA libraries in pooled or arrayed formats are designed to screen genes on a large scale. In arrayed screens, each shRNA is tested in an individual well for the study of biologically subtle and complex phenotypes, which requires the development of protocols for efficient production of high-titer viruses for each shRNA. In pooled formats, screening can be performed efficiently without costly liquid- and plate-handling automation, making them available to more laboratories. In pooled shRNA libraries, thousands of different shRNAs are introduced into a cell population, and these cells are then selected for the phenotype of interest. Pooled shRNA screening is a multi-step process, and each step may affect the quality of the data and the final screening results. Therefore, appropriate screening parameters and careful assay development and optimization are critical. Amerigo Scientific offers high-quality pre-made shRNA libraries targeting whole genome or elite genes as powerful and cost-efficient tools for large-scale loss-of-function screens across diverse biological processes.
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