Quantitative real-time PCR protocol for analysis of nuclear receptor signaling pathways

TLDR

Characterizing tissue expression patterns of nuclear receptors and their target genes across pharmacological conditions and genotypes is essential for understanding their roles. The study aims to provide a standardized, high‑throughput qPCR protocol to quantify transcript levels and changes in nuclear receptor signaling in tissues or cells. The protocol uses reverse‑transcription qPCR to rapidly measure relative transcript abundance and changes across many samples, with forthcoming primer/probe data for the full receptor superfamily.

Abstract

A major goal of the Nuclear Receptor Signaling Atlas (NURSA) is to elucidate the biochemical and physiological roles of nuclear receptors in vivo. Characterizing the tissue expression pattern of individual receptors and their target genes in whole animals under various pharmacological conditions and genotypes is an essential component of this aim. Here we describe a high-throughput quantitative, real-time, reverse-transcription PCR (QPCR) method for the measurement of both the relative level of expression of a particular transcript in a given tissue or cell type, and the relative change in expression of a particular transcript after pharmacologic or genotypic manipulation. This method is provided as a standardized protocol for those in the nuclear receptor field. It is meant to be a simplified, easy to use protocol for the rapid, high-throughput measurement of transcript levels in a large number of samples. A subsequent report will provide validated primer and probe sequence information for the entire mouse and human nuclear receptor superfamily.