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

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.

Figure 1. Typical work-flow for designing and implementing Real-Time PCR assays.

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Figure 2. Representation of RNA priming for reverse transcription.

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Figure 3. Primer set validation.

(a) Example of a valid primer set for mouse LXR- α using SYBR® Green. The presence of a single peak in the dissociation curve and the -3.3 slope and 0.99 R2 value of the standard curve plot are indicative of a good set of primers. (b) Example of an invalid primer set for mouse HSD due to the amplification of non-specific products, as indicated by the presence of multiple peaks in the dissociation curve. This may be an effect of non-specific priming or primer dimerization. Note that the slope of the standard curve plot (-3.2) is within the acceptable range of a valid primer set, but the dissociation curve renders this set of primers unacceptable. (c) Example of an invalid primer set for mouse PNR due to poor amplification. Note the unacceptable slope (-2.28) in the standard curve plot, and the presence of multiple peaks in the dissociation curve.

Figure 4. Formulas for Q-PCR calculations

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Figure 5. Expression profile for mouse LXR- α generated using the standard curve method.

The relative levels of the mRNA transcript are shown (S.D. (from triplicate readings). 18S rRNA was used as the normalizer gene so that the level of LXR- α may be compared between tissue types.

Figure 6. Typical experiment in which wild-type male mice were treated with several different nuclear receptor agonists.

The relative changes in mRNA expression for known receptor target genes, in this case SREBP-1c, an LXR target gene, were measured using the comparative Ct method. (a) SREBP-1c expression plotted for each of the animals (S.D. Each color represents a different experimental group (n=4 animals). (b) SREBP-1c expression plotted as averages of the fold changes for the 4 animals in each treatment group shown in (a) (SEM. Note that the SREBP-1c transcript increases relative to control (VEH) in the animals treated with an RXR agonist (LG268), an LXR agonist (T1317), or both LG269 and T1317 together (T+LG). Note that ligands for PPAR- α (fenofibrate), PPAR- γ (troglitazone), FXR (CDCA), PXR (PCN), and CAR (TCPOBOB) have no effect on the level of the SREBP-1c message relative to control.