Assay Development and Screening
Optimization of Assays for Challenging Targets for Lead Discovery and Screening
The precise regulation of gene expression programs is responsible for the establishment and maintenance of cell, tissue and organ identity, for cellular responses to signaling cues and injuries, and, when disrupted or rewired, for diseases such as cancer and inflammation. Measuring gene expression in high-throughput assays often requires reporter cell line engineering, or using antibodies against endogenous protein markers, which involves a lengthy development process, and can also suffer from batch-to-batch variation. On the other hand, single molecule RNA Fluorescence In Situ Hybridization (smRNA-FISH) detects endogenous transcripts, and is based on DNA oligonucleotide probes that can be rapidly designed in silico, chemically synthetized, tested, and scaled up. For these reasons, smRNA-FISH has the potential to be a useful additional tool for High-Content Imaging (HCI) in chemical or functional genetics screens for the identification of gene expression regulatory pathways. However, visualization of RNA at the single cell level via smRNA-FISH has not been optimized for HCI assays. To address these limitations, we adapted the single-step, enzyme free RNA Hybridization Chain Reaction (RNA HCR) to a 384-well format using an HCI platform. First, we used RNA HCR probes against IFIT3, an interferon stimulated gene (ISG), to demonstrate that high-throughput RNA HCR can quantitatively measure gene expression changes at the single cell level in a 384-well format. As a proof of principle, we performed a focused RNAi screen against 521 human genes involved in epigenetics regulation to identify novel factors mediating the transcriptional response to interferon-γ. The results of this primary screen suggest that multiple components of the MOF acetylase complex are involved in the upregulation of IFIT3 upon interferon stimulation. Finally, we applied high-throughput RNA HCR in other HCI assays to measure expression levels of specific mRNA splicing isoforms of the FGFR2 gene, to monitor the effect of steroid treatment on the expression of inflammation regulators in primary human monocytes, and to determine the effect of steroid treatment on a variety of GR-responsive genes in mouse cells. Altogether, these results indicate that RNA HCR can be miniaturized in 384-well assays to semi-quantitatively detect several endogenous RNA species via HCI in physiologically relevant systems, at the single-cell level, and in a medium- to high-throughput format. In the future, we expect that high-throughput RNA HCR will be useful for the discovery and validation of diverse targets regulating gene expression.