Human Hypoxia Signaling Pathway PCR Array

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Hypoxia Signaling Pathway PCR Array

NOTE: To access content of RT² Profiler PCR Arrays (Prior to May 25, 2012), please click here.
Please NOTE: The RT² Profiler PCR Array System was upgraded to Version 4.0 on May 25, 2012. This system has replaced the Version 3.0 system with regards to content and assay design, with the Version 3.0 products set for discontinuation effective December 1, 2012.

Human Hypoxia Signaling Pathway PCR Array
The Human Hypoxia Signaling Pathway RT² Profiler PCR Array profiles the expression of 84 genes that respond to low oxygen levels. Oxygen is required for aerobic energy metabolism processes such as oxidative phosphorylation. Low oxygen conditions activate the hypoxia signaling pathway in eukaryotic cells, primarily via the hypoxia inducible factor (HIF) transcription factor. HIF heterodimers consist of a constitutively-expressed beta subunit and one of 3 alpha subunit isoforms whose expression is tightly regulated. The presence of oxygen activates prolyl hydroxylases to hydroxylate HIF, leading to its polyubiquitination and degradation. Under low oxygen conditions, prolyl hydroxylase inactivity allows HIF to accumulate, initiating target gene expression. Hypoxia-inducible target genes mediate multiple biological functions, such as angiogenesis, hematopoiesis, and the maintenance of vascular tone to provide or replenish tissues with blood and oxygen. Hypoxia signaling dysregulation commonly occurs in diseases such as tumor angiogenesis and chronic inflammation. Hundreds of HIF target genes have been identified using experimental techniques such as expression studies and chromatin immunoprecipitation (ChIP) as well as bioinformatic analysis of predicted transcription factor binding sites. This array includes HIF signaling transcription factors, HIF interacting proteins, and highly relevant target genes identified by multiple studies. Results obtained with this array can be used to analyze activation or inhibition of hypoxia signaling. Using real-time PCR, research studies can easily and reliably analyze the expression of a focused panel of genes related to the hypoxia signaling pathway with this array. 96-well Plate, 384-well (4 × 96) Plate, and 100-well Disc formats are available. Available for cells, tissues, FFPE samples and small samples	PAHS-032Z-2 PAHS-032Z-12 PAHS-032Z-24 PAHS-032C-2 PAHS-032C-12 PAHS-032C-24 PAHS-032D-2 PAHS-032D-12 PAHS-032D-24 PAHS-032F-2 PAHS-032F-12 PAHS-032F-24 PAHS-032E-4 PAHS-032G-4 Protocol Guide

Functional Gene Grouping

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Reagents & Software

Pathway Source References Modify this Array Gene Table
HIF1 & Co-Transcription Factors: ARNT, COPS5, HIF1A, HIF1AN, HIF3A, HNF4A, NCOA1, PER1. Other HIF1 Interactors: APEX1, EGLN1, EGLN2, NFKB1, P4HA1, P4HB, TP53. Responsive Genes: Angiogenesis: ADORA2B, ANGPTL4, ANXA2, BTG1, EGR1, EDN1, EPO, F3, GPI, HMOX1, JMJD6, LOX, MMP9, PGF, PLAU (uPA), SERPINE1 (PAI-1), VEGFA. Coagulation: ALDOA, ANXA2, F10, F3, F3, PLAU (uPA), SERPINE1 (PAI-1), SLC16A3. DNA Damage Signaling & Repair: ATR, MIF, NDRG1, RUVBL2. Metabolism: ALDOA, DDIT4 (REDD1), ENO1, ERO1L, GBE1, GPI, GYS1, HK2, LDHA, PDK1, PFKFB3, PFKFB4, PFKL, PFKP, PGAM1, PGK1, PKM2, SLC2A1, SLC2A3, TPI1. Regulation of Apoptosis: ADM, BNIP3, BNIP3L, BTG1, DDIT4 (REDD1), IER3, MIF, NOS3 (eNOS), PIM1. Regulation of Cell Proliferation: ADM, BTG1, BLM, CCNG2, EGR1, IGFBP3, MET, MIF, MXI1, NAMPT, NOS3 (eNOS), ODC1, PGF, PIM1, TXNIP. Transcription Factors: BHLHE40, FOS, RBPJ, USF2. Transporters, Channels & Receptors: SLC2A1, SLC2A3, SLC16A3, TFRC, VDAC1. Other Responsive Genes: ANKRD37, CA9, CTSA, DNAJC5, EIF4EBP1, LGALS3, MAP3K1.
Pathway Source References Modify this Array Gene Table

Functional Gene Grouping

How It Works

Manual & Resources

Reagents & Software

"If you have access to a real-time PCR instrument, you can use PCR Arrays"

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 Performance Data Application Data

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

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 25 ng as much as 5 µg of total RNA from a pathway representing genes expressed at a lower level (inflammatory cytokines and receptors).

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).

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Functional Gene Grouping

How It Works

Manual & Resources

Reagents & Software

The complete RT² Profiler™ PCR Array System insures that you will get good results from your pathway-focused gene expression analysis experiment the first time, and every time guaranteed.

The Complete RT² Profiler™ PCR Array System Includes:

RT² Profiler PCR Arrays
Over 100 pathways available
RT² First Strand Kit
Sensitive qRT-PCR with RNA Quality Control and NO Genomic DNA Worries
RT² qPCR Master Mixes
Instrument-specific PCR master mixes insure high-efficiency, gene-specific amplification for the most accurate real-time PCR results.
PCR Array Data Analysis Software (Excel & Web based)

Our convenient and high-quality real-time RT-PCR accessory products complete the RT² Profiler™ PCR Array System and the RT² PCR Primer Assays. The other components needed for real-time PCR analysis are SYBR Green PCR master mix, first strand synthesis kit, and an optional RNA QC array.

Other recommended accessory products

NEW: RT² RNA QC PCR Array
Tests for RNA Quality and Inhibitors of RT-PCR Analyses, optional

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