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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 and transgenic animals. See the complete list of PCR Arrays.
You can use any 96-well or 384-well real-time PCR instrument. See
instrument-specific setup instructions. Click below for detailed
information.
How it Works
The PCR array is a set of optimized real-time PCR primer assays on 96-well or
384-well plates 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 plate, and then run the real-time PCR
cycling program. (Download user manual)
| Figure 1: |
How PCR Arrays Work - Protocol Chart |
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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 both 96- and
384-well plates 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 Nano 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. |
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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.
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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).
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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.
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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.
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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).
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