High-throughput, amplification-free detection of endogenous gene expression perturbations via fluorescence anisotropy
摘要
High-throughput detection of gene expression changes enables novel biological discovery. However, current methods suffer limitations. Methods coupled with RNA-sequencing involve higher costs and technological barriers while gene reporter assays only account for promoter activity. Here, 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 expression changes of endogenously expressed genes with potential for duplex measurements and can quantify absolute numbers of transcripts. In a high-throughput chemical screen for oncogene MYC expression modulators, CFAR identified hits more robustly than the traditional gene reporter assay. From the screen, 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.
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