High-throughput, amplification-free detection of endogenous gene expression perturbations via fluorescence anisotropy.

High-throughput detection of gene expression changes enables novel biological discovery. However, current methods coupled with RNA sequencing involve higher costs and technological barriers, while gene reporter assays account for only promoter activity. We developed a high-throughput, amplification-free, and accessible method-cellular fluorescence anisotropy of RNA (CFAR)-for assessing the effect of multiple perturbations on the endogenous expression of one or a few genes. CFAR measures cellular RNA levels through changes in fluorescence anisotropy of fluorescent dye-labeled DNA probes. Heating and centrifugation steps remove unwanted cellular components. CFAR robustly detects relative expression changes of endogenously expressed genes with duplex measurements. In a high-throughput chemical screen for oncogene MYC expression modulators, CFAR identified hits more robustly than the traditional gene reporter assay. We validated FDA-approved drugs that can potentially be repurposed for downregulating MYC expression in colorectal cancer cells. CFAR is an accessible alternative tool for assessing perturbations on endogenous gene expression.