Human IL6 Quantitative mRNA Plate Assay

Interleukin 6 (IL-6), which was originally identified as a B-cell differentiation factor, is now known to be a multifunctional cytokine that regulates the immune response, hematopoiesis, the acute phase response, and inflammation. It is produced by activated T cells, macrophages, endothelial cells, fibroblasts and osteoblasts. Deregulation of IL-6 production is implicated in the pathology of several disease processes, such as autoimmune diseases and chronic inflammatory proliferative diseases. Monitoring the expression of this cytokine is therefore a common approach to dissect the molecular mechanism underlying the diseases. 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 IL6 quantitative mRNA plate assay, which can be used for quantitative measurement of IL6 gene expression directly in cell lysates as well in total RNA.

A. Benefits:

Signosis' quantitative mRNA plate assay offers following benefits:

• Absolute Quantitative Assay - IL6 mRNA can be quantitatively measured using IL-6 in vitro RNA as a control.

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

• Simple -Cell lysates or total RNA can be directly applied to plate hybridization. It does not need total RNA preparation, and 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.

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