High-throughput screening for RNA interference
By Dr. Afsaneh Motamed-Khorasani

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RNA interference (RNAi) involves the knockdown of specific gene functions. It is a reverse genetic approach for the functional analysis of a large number of genes. Furthermore, it offers the identification of structure or function of the genes, relevant to specific pathway by gene knockdown mechanisms.

RNAi screening is also used for better understanding of host-pathogen interaction and cancer biology. RNAi screening has been automated for high-throughput drug discovery studies and requires a good knowledge of computer science and engineering for automated high-content image acquisition and analysis.

Continuous improvements of the RNAi screening have been achieved over the past few years through performing a large number of screens and the detailed inputs of RNAi pathways and mechanisms. Cell-based RNAi screening could lead to a better understanding of the functional genomics in human cells as well as other cell types.

Recently, cell-based RNAi screenings have been performed in the neuronal and muscular primary cells, haemocytes of drosophila and mammalian stem cells. RNAi screen data has presented valuable information about the cell signaling and single-nucleotide polymorphism (SNP).

A high-throughput RNAi screening was recently developed to identify the novel regulators of the internal ribosome entry site (IRES)-driven mRNA translation in human cells. In this approach, researchers combined the conventional RNAi screening method with an automated transfection of a reporter mRNA of interest in order to improve its performance.

This robust method could be used for the quantitative analysis of the IRES-dependent translation as well as its comparison with the conventional cap-dependent translation activity using monocistronic reporter mRNAs. MAPK3 kinase, a novel positive regulator of vascular endothelial growth factor-IRES-driven translation, was discovered by using this novel method. The method was also scalable and could be adapted for a high-throughput identification of the regulators of mRNA translation.

The arrayed RNAi screening of the cells could be conducted by varying RNAi reagents in a multiwell plate and analyzing the reagent conditions by readout machines. The RNAi reagents are printed on microarray slides to facilitate the reverse transfection of the cells. Microfluidics and microarray plates might be the next frontiers in the arrayed RNAi screening.

RNAi screening could be combined with different types of readouts. Resolution at the individual cell level is achieved by using a fluorescence activated cell sorter (FACS) in the array-based screens, and the subcellular based information is determined by fluorescent labeled dyes, probes or antibodies. By using software such as CellProfiler, hundreds of screen-image data sets could be analyzed to identify the phenotypes relevant to the topic of interest.

Pooled RNAi screening is used for screening large RNAi reagent collections with the goal of introducing one RNAi reagent per cell. Pooled screen readouts could be used to compare two or more population of cells for a better understanding of various mechanisms such as cancer. However, the interpretation of the pooled screen results could be challenging. Recently a microarray-based technology was developed for the deconvolution of the pooled shRNA screens with 90,000 shRNAs.

Another innovation in the field of RNAi screening has been the double-knockdown screens. It involves large-scale combined targeting of more than one gene. This technique addresses the issues of redundancy in genetic networks. Large-scale RNAi screens in drosophila have provided valuable information about the redundancy and connectivity of the conserved signal transduction pathways.

Although cell-based RNAi screening provides insights into understanding the phenotype of a condition, more complex phenotypes in a particular tissue or stage of an individual could be determined by in vivo RNAi screening. RNAi-based libraries have been developed for many species, including: lepidoptera and other insects, many types of ticks, hydra, planarians, a variety of plants, and pathogens such as trypanosomes. In vivo RNAi screens have been also developed for C.elegans, drosophila, and mice.

In conclusion, high-throughput RNAi screening is a power tool to better understand gene function, cell networks and biomedicine. It is particularly useful to understand cancer biology and host-pathogen interactions. Arrayed cell-based screens could provide information with the help of sophisticated assay analysis instruments. In vivo RNAi screening of different animals provides a holistic approach about diseases like obesity and aging. The scope and quality of the RNAi screens needs to be improved in the future in order to address more diverse problems.

Dr. Afsaneh Motamed-Khorasani is a medical and scientific affairs specialist with a strong background in biomedical sciences, clinical trial/research and medical/regulatory writing/submission. She is the president and managing director of Neometrix Consulting Inc., which helps global pharmaceutical and medical device companies with their medical and regulatory writing and submissions as well as medical affairs.