Human HIF-Regulated cDNA Profiling in Cell Lysates

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). Through coupling its CL(TM) cDNA synthesis kit which allows reverse transcription directly in cell lysates without RNA preparation, Signosis has developed a plate-based hybridization array for profiling the expression of 20+ HIF-regulated genes directly in cell lysates.

A. Benefits:

HIF-regulated cDNA profiling in cell lysates offers following benefits:

• Simple - cDNA synthesis directly in cell lysates without RNA preparation. In addition, the detection is as simple as ELISA.

• High sensitive detection- Detected mRNA molecules are converted into multiple biotin-labeled cDNAs for sensitive detection.

• Quantitative comparison – The difference of two or more samples in gene expression can be quantitatively analyzed.

B. Principle of the technology

Unlike conventional reverse transcription which needs total RNA, Signosis has developed a CL(TM) cDNA synthesis-based plate array. It can synthesize cDNA probes through reverse transcription directly in cell lysates without RNA preparation. The synthesized cDNA probes are then applied for a plate array hybridization. Targeted genes are specifically captured onto individual wells on a plate 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.

Home |  Product List |  Literature |  Order |  Contact Us