Gene Expression Kits & Services

Knowing that your compounds regulate the activity of a specific nuclear receptor using INDIGO’s services are a great first step in characterizing and prioritizing potential new drugs. However, this is just a beginning to fully understanding the biologic (or toxicologic) effects that may ensue. Gene expression is a widely used approach for characterizing biological perturbations, defining the molecular mechanisms of diseases and making critical decisions about risks associated with compounds of interest.

Quantitative real time PCR (qPCR) is the gold standard for gene expression analysis. Our team of experts in nuclear receptor biology and toxicology will provide the know-how in gene selection, primer design and mRNA accumulation analysis.

Real-time PCR is a variation of the standard PCR technique used to quantify DNA or RNA in a sample. Using sequence-specific primers, the relative number of copies of a particular DNA or RNA sequence can be determined. By measuring the amount of amplified product at each stage during the PCR cycle, quantification is possible. If a particular sequence (DNA or RNA) is abundant in the sample, amplification is observed in earlier cycles; if the sequence is scarce, amplification is observed in later cycles.

Quantification of amplified product is obtained using fluorescent probes or fluorescent DNA binding dyes and real-time PCR instruments that measure fluorescence while performing temperature changes needed for the PCR cycles. Standard curves were made using serial dilutions from pooled cDNA samples. Real Time PCR was performed using the SYBR Green PCR Mater Mix according to the manufacturer’s protocol and amplified on the ABI Prism 7300 Sequence Detection system.

QPCR Solutions:

• INDIGO offers predesigned panels of optimized primers for nuclear receptor target genes including PXR, CAR, and PPARs.
• Experience in gene selection for disease and pathway-specific studies including inflammation-, cancer-, diabetes-, and drug metabolism-related gene expression.
• We will provide data on mRNA levels relative to a housekeeping gene, statistical analysis and interpretation in our study report.

INDIGO also provides comprehensive DNA microarray solutions, using commercial offerings from Affymetrix, for use in biomarker discovery, gene ontology analysis and predictive toxicology. All array services include quality control of array performance data and statistical, clustering and pathway analysis needs.

Microarray Solutions:

• INDIGO offers the Affylmetrix microarray platform for comprehensive analysis of gene expression in human, rat and mouse tissues.
• In addition to providing support for purchase of the appropriate array for your study, we will perform RNA extraction, cDNA labeleing, RNA quality control, array hybridization and data collection.
• Our suite of data analysis includes generating lists of genes significantly regulated under your treatment conditions as well as gene ontology and pathway analysis.

Atherosclerotic plaques contain macrophages that have ingested large amounts of cholesteryl ester, forming lipid droplets and gaining the appearance of a “foam cell.” During the process of reverse cholesterol transport (RCT), excess peripheral cholesterol is scavenged by tissue macrophages, which process cholesterol and transport it to the liver via HDL for excretion. In macrophage, control of the initial steps of RCT (cholesterol uptake and efflux) is manifested by a variety of NRs, in particular the PPARs and LXRs. As part of INDIGO’s RCT assay, THP-1 cells are converted to foam cells upon treatment with oxidized LDL and are loaded with a fluorescent cholesterol derivative. Subsequently, the effects of clients’ compounds of interest are examined for their ability to induce the transport of fluorescent cholesterol to HDL.

Historically, primary hepatocytes have been the preferred in vitro model for assessing drug-induced expression of drug metabolizing enzymes. However, their limited supply from any one donor and their finite life span pose challenges to their routine use for iterative, comparative drug screening.

Immortalized human heptoma (i.e., HepG2) and hepato-carcinoma cell lines are sometimes used owing to their unlimited proliferative potential. However, a serious limitation to using transformed cell lines is their decreased, or absent, expression of hepatocyte differentiation markers. These include nuclear receptors and other xenobiotic-sensing receptors and, hence, their target genes (e.g., CYPs) whose expression are critical to correctly assessing the potential liability of induced drug-drug interactions.

INDIGO's assay kit for the Expression Profiling of Clinically Relevant CYPs utilizes upcyte^® hepatocytes, which are human donor-derived hepatocytes established by upcyte technologies GmbH. These cells have the attributes of limited proliferation while maintaining their native high levels of constitutive and inducible xenobiotic metabolizing enzyme activities. Like primary hepatocytes, confluent cultures of upcyte^® hepatocytes express liver-specific proteins, produce urea, and store glycogen. Importantly, the induction profiles of cytochrome p450 (CYP) enzyme activities are similar to those of the primary hepatocytes. Thus, upcyte^® hepatocytes combine the characteristics and advantages of primary hepatocytes with the added practical advantage of having access to the same donor cells for use in iterative, large-scale experiments over extended periods.

For more information on INDIGO's utilization of upcyte^® hepatocytes, view our In Vitro Toxicology Platform.

Cytochrome p450 (CYP) enzymes are responsible for the Phase I metabolism of most drugs. And, it is noteworthy that the expression of cytochrome p450 (CYP) genes are predominantly regulated by ligand-activated receptors/transcription factors such as pregnane X receptor (PXR, NR1I2), constitutive androstane receptor (CAR, NR1I3), aryl hydrocarbon receptor (AhR), farnesoid x receptor (FXR, NR1H4), glucocorticoid receptor (GR, NR3C1), and to lesser degrees liver X receptors (LXR, NR1C3), vitamin D receptor (VDR, NR1I1), and peroxisome proliferator-activated receptor alpha (PPARα, NR1C1). Consequently, drugs that activate any of these xenobiotic sensing receptors can dramatically change the endogenous levels of CYP expression in the liver, potentially impacting the rate of their own metabolism, as well as the metabolism of all other co-administered drugs. Of particular concern are metabolic outcomes that transform a drug to greater potency, or to an altered bioactivity.

Assessing drug-induced changes in the expression of CYP genes provides a reliable predictive indicator of altered (either heightened or inhibited) metabolic activities leading to drug-drug interactions in vivo. Cytochrome p450 enzymes with the greatest clinical relevance belong to CYP1, 2, and 3 families. It is estimated that they are involved in the metabolism of 70-80% of drugs currently on the market. For example, CYP3A4 is estimated to be involved in ~30% of all prescription drugs.

INDIGO's assay kit for the Expression Profiling of Clinically Relevant CYPs contains optimized reagents for the culturing and treatment of upcyte^® hepatocytes to assess drug-induced changes in seven clinically-relevant CYPs: CYP3A4. CYP1A1, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP 2E1.