Characterization
Description(s):Rapidly growing tumors result in hypoxic regions. Adaptive responses of most cells to hypoxia are (1) to produce VEGF and other hypoxia-induced angiogenic cytokines that promote increased tissue vascularization, thereby increasing tissue oxygenation, and (2) to switch metabolically from oxidative phosphorylation to anaerobic glycolysis. Hypoxia-inducible factor 1 (HIF-1) is an oxygen-regulated transcriptional activator that plays essential roles in the process. The HIF-1alpha subunit is oxygen-dependent ubiquitination and proteasomal degradation. In addition, cytokines including interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) stimulate HIF-1 dependent gene expression. HIF-1 increases the expression of several genes that promote blood flow and inflammation, such as vascular endothelial growth factor (VEGF).
Signosis developed a plate-based array for profiling 20+ HIF-regulated genes. By using this assay, researchers are able to screen up to three samples on a single microtiter plate to compare gene expression in three separate constructs side by side.
Applicable Grid:
List of Applicable Genes
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| A | ANGPTL4 | Glut-3 | PDGF-A | ANGPTL4 | Glut-3 | PDGF-A | ANGPTL4 | Glut-3 | PDGF-A | ANGPTL4 | Glut-3 | PDGF-A |
| B | 18SrRNA | HIF-1a | PDK1 | 18SrRNA | HIF-1a | PDK1 | 18SrRNA | HIF-1a | PDK1 | 18SrRNA | HIF-1a | PDK1 |
| C | BCL2 | HIF-2a | RTP | BCL2 | HIF-2a | RTP | BCL2 | HIF-2a | RTP | BCL2 | HIF-2a | RTP |
| D | CREBP | HPRT | SAG | CREBP | HPRT | SAG | CREBP | HPRT | SAG | CREBP | HPRT | SAG |
| E | CTSD | IGF-II | UCP3 | CTSD | IGF-II | UCP3 | CTSD | IGF-II | UCP3 | CTSD | IGF-II | UCP3 |
| F | EP300 | Leptin | VEGF | EP300 | Leptin | VEGF | EP300 | Leptin | VEGF | EP300 | Leptin | VEGF |
| G | EPO | MMP2 | VHL | EPO | MMP2 | VHL | EPO | MMP2 | VHL | EPO | MMP2 | VHL |
| H | Glut-1 | PAI-1 | Blank | Glut-1 | PAI-1 | Blank | Glut-1 | PAI-1 | Blank | Glut-1 | PAI-1 | Blank |
Principle
Signosis’ proprietary cDNA plate array is a plate-based hybridization profiling analysis for monitoring the expression of dozens of genes through reverse transcription of mRNA into cDNA. Like array analyses, total RNA is first reverse transcribed into cDNA in the presence of biotin-dUTP in the assay. Targeted genes are then specifically captured onto individual wells on a plate, instead of membranes, through a pre-coated gene-specific oligonucleotide. The captured cDNAs are further detected with streptavidin-HRP. Luminescence is reported as relative light units (RLUs) on a microplate luminometer. The expression level of genes is directly proportional to the luminescent intensity.
Data
Mouse HIF-regulated cDNA plate array analysis. NIH3T3 cells were treated with and without 200 uM cobalt chloride for 24 hours, RNAs prepared, cDNA synthesized with biotin label and subjected to cDNA plate array hybridization and detection.
