Human TNF Quantitative mRNA Plate Assay

Tumor necrosis factor-alpha (TNF-a) is one of inflammatory cytokines. It plays a variety of functions, although many of them are not yet fully understood. It is produced by different types of cells, but especially by macrophage. Altered expression of inflammatory cytokines including TNF-a has been shown to associate with different diseases, such as inflammatory bowl disease. Monitoring the expression of these cytokines is therefore a common approach to dissect the molecular mechanism underlying he diseases. In addition, the emergence of molecular medicine has resulted in the desire of quantitative analysis of these molecules in clinical diagnostics. Signosis has developed a multiple-biotin signal amplification (MBSA) technology. It is 1000 times more sensitive than single biotin-based detection. Based on this technology, Signosis developed TNF-a quantitative mRNA plate assay. It can absolutely measure human TNF-a mRNA in lower fetogram range.

A. Benefits:

Signosis' quantitative mRNA plate assay offers following benefits:

  • Absolute Quantitative Assay - TNF mRNA can be quantitatively measured using TNF in vitro RNA as a control.

  • Higher sensitivity - The assay can measure as few as 500 molecules with a broader dynamic range.

  • Simple - Total RNA is directly applied to plate hybridization. It does not need enzymatic conversion from mRNA into cDNA. The entire assay procedure is “incubation and wash”.

B. Principle of the technology

In MBSA, multiple biotins are added to a polymer, which contains a tag complementary sequence that can hybridize with a linker sequence containing both a target sequence and a tag sequence. A stretch of linker sequence shares a common tag sequence, although they contain different target sequences to hybridize different regions of a target gene. Therefore, two-layer amplification can be achieved; multiple regions and multiple biotins. RNA plate assay is integrated with MBSA, in which a target mRNA is captured onto a plate with a number of capture oligos. The captured RNA molecule was also hybridized with multiple linkers, which target different regions of the mRNA. MBSA is then hybridized with the linkers. The captured biotin molecule is detected with streptavidin-HRP and a chemiluminescent substrate.

Figure 1
Diagram

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Figure 2. TNF IVT RNA standard curve (A) and measurement of TNF induction in U937 (B & C). TNF IVT was serially diluted and subjected to TNF quantitative RNA plate assay (A). U937 cells were treated with and without 50ng/ml LPS for 2 and 4 hours. 1ug of total RNA was used for TNF quantitative mRNA plate assay for TNF expression (B). GAPDH is used for normalization (C).

 

 Product Size Cat. Price
Human TNF Quantitative mRNA Plate Assay 96 RA-0001 $459
  • Human TNF Quantitative mRNA Plate Assay User Manual (PDF)

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