Quantitative PCR-based Assay to Measure Sonic Hedgehog Signaling in Cellular Model of Ciliogenesis.

Abstract

Ciliopathy refers to a collection of multisystemic and polygenic human diseases and disorders that arise due to aberration of structure or function of motile or non-motile (primary) cilia that are microtubule-based cell protrusions. Primary cilia (PC) are present in most human epithelial and endothelial tissues and serve as a hub for physiological signal transduction, including Sonic Hedgehog (SHH) signaling. Various situations demand examining the function of PC, which may be achieved by measuring the canonical SHH signaling pathway that is almost exclusively mediated by PC. Here, a quantitative PCR (qPCR) based technique is developed to measure the transcriptional level of SHH pathway genes in quiescent hTERT-RPE1 (or RPE1) cells that mostly contain PC. Quiescence in RPE1 cells is achieved by serum starvation for 48 h, while activation of the SHH pathway is promoted by a specific agonist. This cell culture-based assay is easy to follow and sensitive. Successful demonstration of this assay in ciliated RPE1 cells indicates its immense potential as an in vitro assay to examine the proper function of PC upon genetic or epigenetic alteration of one or multiple genes, which may be associated with various ciliopathies.