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RT² Profiler™ PCR Array System Application Example

PCR Arrays have been increasingly used in all areas of life science and biomedical research including:

  • Cancer
  • Inflammation and cytokine profiling
  • Stem cells
  • Neuroscience
  • Signal transduction pathways
  • Cell adhesion and cell migration
  • Biomarker screening and validation

We list below a few examples of application data generated by our R&D group. To see the research using PCR Arrays published by the scientific community, please see our Publication List.

Cancer Research:
Gene expression profiling is important for discovering and validating tumor biomarkers and therapeutic targets. Using the Cancer PathwayFinder PCR Array and the Human Extracellular Matrix and Adhesion Molecules PCR Array, we examined the gene expression profiles exhibited by two different human breast tumors relative to normal tissues. The study compared the relative expression of both tumorigenesis- and adhesion-related genes between each tumor sample and a normal breast tissue sample. This study provides an example of the identification of a pathway affected by the transformation of a particular tumor type.

Template cDNAs prepared from total RNA of normal human breast and two human breast tumors (BioChain Institute, Inc., 5.0 µg) were characterized in technical triplicates using the Human Cancer PathwayFinder™ PCR Array and the Human Extracellular Matrix & Adhesion Molecule PCR Array with the RT² SYBR Green / Fluorescein PCR master mix on the Bio-Rad iCycler®

Figure 1: ECM & Adhesion PCR Arrays Revealed Up- and Down-Regulated Genes in Breast Cancer
Total RNA from normal human breast and a human breast tumor were characterized in technical triplicates, and the relative expression levels for each gene in the two samples are plotted against each other in the Scatter Plot. Genes encoding the matrix metallopeptidases (MMP3 & MMP9) and their inhibitors (TIMP3) are up-regulated, while genes encoding integrins (ITGB3 & ITGB4) are down-regulated, by at least three-fold (outside the silver field) in breast tumors relative to normal tissue.

Toxicology Research:
Rezulin (Troglitazone or "Tro" or "T"), a glitazone PPAR-gamma agonist, was approved for treatment of type 2 diabetes mellitus, but was withdrawn from the market due to idiosyncratic liver toxicity. Two similar drugs, Avandia (Rosiglitazone or "Rosi" or "R") and Actos (Pioglitazone or "Pio" or "P"), are considered to be safe treatments for the same condition. The expression profile of key drug metabolism genes should be different in cells treated with Rezulin versus those treated with Avandia and Actos.
Hepatocellular carcinoma HepG2 cells were treated at 80% cell confluence with these three drugs (100 µM, Cayman Chemical) or a DMSO vehicle control for 24 h. RNA isolated using the ArrayGrade™ Total RNA Isolation Kit was used to characterize gene expression with the Human Drug Metabolism and Stress & Toxicity PathwayFinder™ RT² Profiler™ PCR Arrays and RT² SYBR Green / Fluorescein PCR master mix on the Bio-Rad iCycler®

Figure 2: Stress & Toxicity PathwayFinder PCR Array Uncovered Distinct Gene Expression Profiles Associated with Liver Toxicity Caused by 3 PPARg Agonists (Actos, Avandia, and Rezulin)
RNA from HepG2 cells treated with three different glitazone PPAR? agonists for type 2 diabetes mellitus was characterized, and the results were compared to that of a vehicle (DMSO) control. A withdrawn drug with idiosyncratic liver toxicity (Rezulin) induces very different changes in the expression of stress-related genes than two safer drugs still on the market (Avandia and Actos).

Cytokine Profiling
Cytokine quantification is an important element in studies of inflammation and immune responses. Quantitative RT-PCR, a rapid and sensitive assay, is the preferred method to quantify cytokine mRNA levels because they are often expressed at low levels. The PCR Array System offers a simple, reliable and sensitive tool for multiple cytokine profiling. Using the Human Cytokine PCR Array, we have monitored the mRNA levels of 84 different cytokines in stimulated versus and untreated human peripheral blood mononuclear cells (PBMC).

Figure 3: Common Cytokine PCR Array Identified 23 Up-Regulated and 6 Down-Regulated Genes Following PMA and Ionomycin Treatment.
Triplicate total RNA samples from human peripheral blood mononuclear cells (either untreated or stimulated with 50 ng/mL PMA and 1 mg/mL ionomycin for 6 hours) were characterized. Twenty-three cytokine genes are up-regulated (> 5-fold, p < 0.0005) including interleukins, colony stimulating factors, and TNF ligands after 6 hours of stimulation. Six interleukin and TNF ligand genes are down-regulated under the same conditions.