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How It Works: RT² Profiler™ PCR Array System

RT² Profiler™ PCR Arrays are the most reliable and sensitive gene expression profiling technology for analyzing a panel of genes in signal transduction pathways, biological process or disease related gene networks. The PCR Arrays can be used for research on cancer, immunology, stem cells, toxicology, biomarker discovery and validation, and phenotypic analysis of cells, tissues, and research animal samples. See the complete list of PCR Arrays.

You can use any 96-well plate, 384-well plate, or 100-well disc real-time PCR instrument. See instrument-specific setup instructions. Click below for detailed information.

How it Works Performance Data Application Data

How it Works

The PCR Arrays are sets of optimized real-time PCR primer assays on 96-well plate, 384-well plate, or 100-well disc for pathway or disease focused genes as well as appropriate RNA quality controls. The PCR array performs gene expression analysis with real-time PCR sensitivity and the multi-gene profiling capability of a microarray. Simply mix your cDNA template with the appropriate ready-to-use PCR master mix, aliquot equal volumes to each well of the same PCR Array, and then run the real-time PCR cycling program. (Download user manual)

Figure 1: How PCR Arrays Work - Protocol Chart

What it offers?
     Guaranteed Performance* - ready-to-use for gene expression analysis
     Time and cost saving - less than 30 min hands-on time for analyzing 84 genes
     Ease of data analysis using our easy-to-use Excel-based data analysis template or web-based analysis tool

Layout and Controls: The PCR Arrays are available in 96-well plate, 384-well plate, and 100-well disc formats, and are used to monitor the expression of 84 genes related to a disease state or pathway plus five housekeeping genes. Controls are also included on each array for genomic DNA contamination, RNA quality, and general PCR performance.

You can easily perform a PCR Array experiment in your own laboratory, or send your samples to us and take advantage of our PCR Array Services.

*: when using complete PCR array system.

Performance Data Sensitivity:
The complete PCR Array System yields a greater-than 85 percent present call with as little as 1 ng as much as 5 µg of total RNA from a pathway representing genes expressed at a lower level (inflammatory cytokines and receptors). 1ng total RNA sensitivity is possible when RT² PreAMP Kits are used.


Figure 2: PCR Arrays Let You See More Genes with Less RNA
Different amounts of universal total RNA were characterized using the Human Inflammatory Cytokines and Receptors PCR Array, and the percentage of detectable genes was calculated for each RNA amount.
As little as 25 ng total RNA yields greater than an 80% positive call, even for cytokines expressed at very low levels.

Reproducibility
The complete PCR Array System demonstrates strong correlations across technical replicates, lots, and instruments with average correlation coefficients > 0.99 insuring reliable detection of differences in expression between biological samples.

  050825_1  050825_2  050825_3  050825_4
060111_1 0.993 0.989 0.995 0.992
060111_2 0.994 0.990 0.995 0.992
060111_3 0.992 0.990 0.993 0.992
060111_4 0.993 0.992 0.994 0.992
Figure 3: PCR Arrays Yield Highly Reproducible Results
Four replicate sets of raw threshold data (1-4) obtained by two different scientists (A & B) at two different times (050825 & 060111) on Human Drug Metabolism RT² Profiler PCR Arrays are directly compared. The results demonstrate a high degree of correlation (R2 > 0.990).

Specificity
The complete PCR Array System, with high quality input RNA, is guaranteed to yield single bands of the predicted size without primer dimers or other secondary products thus providing the most accurate real-time PCR results possible.

Figure 4: PCR Arrays Amplify A Single Gene-Specific Product in Every Reaction.
Universal total RNA was characterized on the TGFβ / BMP Signaling Pathway PCR Array, followed by dissociation (melt) curve analysis. PCR Arrays specifically detect individual genes despite the expression of related gene family members in the same RNA sample.

Application Data

Cancer Research:
To ascertain the oncogenic route that two different human breast tumors have taken, the relative expression level of cancer- and adhesion-related genes in normal and two different cancerous tissues were compared.

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 5: 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 6: Stress & Toxicity PathwayFinder™ PCR Array Uncovered Idiosyncratic Mechanisms of Action for Liver Toxicity Caused by 3 PPARγ Agonists.
RNA from HepG2 cells treated with three different glitazone PPARγ 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).