Gene Expression Profiling

INDIGO’s assays are not binding assays. They are cell-based trans-activation assays, and the principal application is in the screening of test samples to quantify any functional activity, either agonist or antagonist, that the compounds may exert against the nuclear receptors. INDIGO reporter systems utilize firefly luciferase reporter gene technology, and while there is a binding taking place, our assays do not measure it. Instead, the luciferase light response is measured which correlates to the activation status of the receptor (either activation or inhibition).

Quantifying changes in luciferase expression in the treated reporter cells provides a sensitive surrogate measure of the changes in receptor activity. It will tell the scientist the significance (strength) of the interaction by the level of light emitted. In addition, cell-based assays are more sensitive and able to detect smaller levels of activation.

INDIGO’s nuclear receptor assays utilize proprietary human and non-human mammalian cells engineered to provide constitutive, high-level expression of the designated receptor. Specific cell type information for each assay is proprietary and available only through consultation with INDIGO’s technical team following a screening service or assay kit purchase.

Reporter cells included in INIDIGO’s steroid hormone nuclear assay kits (ERα, ERβ, AR, PGR, MR, GR) express the native, full-length receptors. INDIGO’s other nuclear receptor assays, however, include reporter cells that express hybrid nuclear receptors. In these cases, the respective receptor’s native N-terminal sequence comprising the DNA Binding Domain (DBD) has been replaced with sequence encoding the yeast Gal4-DBD. All other native NR functional/structural domains (ligand binding domain, hinge region, and various activation domains) are present in these hybrid receptors. These reporter cells also contain the firefly luciferase reporter gene functionally linked to the upstream genetic response element for Gal4.

Consequently, once a bioactive compound associates with the ligand binding domain of the hybrid receptor, only the luciferase reporter gene is induced. Ligand-activation of the hybrid receptor will not induce collateral expression of target genes that are otherwise regulated by the native nuclear receptor.

View our list of reference compounds. We do utilize commercially available reference agonists for our assays, and this reference agonist is included in each assay kit. We do use reference antagonists where a reference is known and commercially available. Some of our antagonist assays do not have reference antagonists available.

Treatment concentrations are prepared at INDIGO using serial dilutions in fixed increments; the starting concentration and increment of dilution is specified by the customer via our Study Work Order sheet that accompanies all screening studies. For accurate determination of EC50 and/or IC50 values, we recommend at minimum spanning a 5,000-fold concentration range over 8 doses. This strategy requires a 3.33-fold or 4-fold increment of serial dilution. The minimum doses recommended for EC50 and/or IC50 value is 7 test concentrations.

Upon completion of your study, you will receive a detailed study report (PDF format) including all assay methods and validation, the raw data and calculations of your study (Excel format), and graphing files of all data (GraphPad Prism format). In addition, you are always welcome to contact INDIGO’s team for assistance in interpretation of your data.