Identification of genes involved in autophagy by RNAi and high throughput imaging
A whole genome siRNA screen is enabled by the IN Cell Analyzer 6000
Whole genome RNAi screening
RNAi screening is used to identify genes involved in cellular biochemical pathways, or processes such as metastatic invasion, differentiation or a myriad of other functions. By systematically knocking down each gene in a gene family, known pathway or across the entire genome and then observing the functional result, genes that enhance or inhibit a cellular process can be identified.
This application note describes an experimental workflow for identification of genes involved in a cellular process known as autophagy – a process by which material in the cytosol is collected into double membrane structures (omegasomes) and then degraded in the lysosome. A cell line in which omegasome formation can be observed was developed by the fusion of a green fluorescent protein (GFP) with a component of the omegasome. In this way, a phenotypic readout for omegasome formation can be observed by fluorescent microscopy.
About this experiment
The goal of this experiment was to identify genes that enhanced or inhibited omegasome formation and are thereby implicated in autophagy. To accomplish this experimental goal, siRNA was used to interrogate 18,480 genes one-by-one in 96 well plates using omegasome formation as the readout.
High throughput imaging
High throughput imaging was used to count omegasome formation. A typical limitation of high content screening platforms is a loss of focus sufficient to resolve intracellular vesicular structures as a system moves from well to well. A second limitation, especially with large scale screens, is the time required to scan and collect images and subsequently process the data. The advanced capabilities of the IN Cell Analyzer 6000 provided efficient data throughput and improved image quality with less well-to-well variation to enable the completion of this siRNA screen.
The findings of this work identified 181 candidate genes from the original 18,480 genes that will be further evaluated to determine importance of their role in autophagy.
The details of this work can be found in this Application Note:
- Practicalities of using a SMARTpool siRNA library to carry out a full genome screen utilizing DFCP1 as a marker of autophagy initiation and the IN Cell Analyzer 6000 for high throughput imaging.
- Dharmacon siRNA products are the result of scientific innovation in siRNA design and novel modification strategies to optimize potency, specificity and delivery.
- What is siRNA and how do you use it?
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