Gene Expression
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.
Whether your laboratory provides its own samples or you allow INDIGO’s experts to implement the study in our labs, we have a gene expression solution for you. Our highly-trained specialists provide a comprehensive offering of genomic services including RNA extraction, qPCR services, microarray services, and microarray data analysis. We will help you identify the appropriate technology, experimental design and program goals to ensure that your needs will be met.
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.