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 has developed a plate-based array for profiling 20+ HIF-regulated genes. By using this assay, researchers are able to compare gene expression in up to three samples on a single microtiter plate.
List of Applicable Genes
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.
Human HIF-regulated cDNA plate array analysis. HeLa 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.
LiteratureView user manual
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3.HIF-stabilizing bioactive glasses for directing MSC behaviour . MM Azevedo, O Tsigkou, R Nair, G Jell, J Jones… - Tissue Engineering, 2014