While quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) is a standard tool for many laboratories studying gene regulation, it is not commonly used for small molecule screening. More commonly, high throughput screens (HTSs) designed to detect transcriptional changes use a gene reporter, such as green fluorescent protein (GFP), β-galactosidase, or luciferase. The downsides of this approach include the genetic manipulation required to make reporter lines, the artifacts introduced by this indirect measurement, and the limited number of genes that can be monitored. Here we describe a method for using qRT-PCR to assay the regulation of multiple genes in a 384-well format. We envision this technology being utilized in three main scenarios: screening against cell lines that are not amenable to genetic manipulation (such as lines derived from patients), screening for transcriptional regulators without well-defined functions, or as a secondary screen validating results obtained using a traditional reporter cell line or biochemical readout. Additionally, we provide useful guidelines and protocols for culturing and plating mouse embryonic stem (ES) cells for high throughput screening. While embryonic stem cells are of great interest for regenerative medicine and are a useful tool for studying the epigenetic regulation of cell identity, they are difficult to culture and require extra care and consideration. We provide a method for culturing and plating mouse ES cells in 384-well format.
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