miR-17-92 Cluster Plate Assay

A polycistronic miRNA cluster miR-17-92 play a role in the control of cell proliferation and angiogenesis. This cluster consists of seven miRNAs; miR-17-5p, miR-17-3p, miR-18a, miR-19a, miR-19b, miR-20a, and miR-92. Like E2F1 gene, the cluster is transcriptionally activated by the proto-oncogene c-Myc. Because two of the expressed miRNAs, miR-17-5p and miR-20a, repress translation of E2F1, the level of E2F1 protein is modulated by this feedback mechanism. Therefore, the accumulation of excessive amounts of E2F1 can be prevented and the states of cells towards apoptosis or cell division can be controlled. In addition, c-MYC-mediated induction of the miR-17-92 cluster results in down-regulation of the anti-angiogenic thrombospondin-1 and related proteins, such as connective tissue growth factor.2, and therefore it promotes angiogenesis. Furthermore, a recent study indicates that the cluster is significantly up-regulated at the clonal expansion stage of adipocyte differentiation. The miR-17-92 cluster has been implicated as oncogenes in numerous tumor types. Effective monitoring of the expression of the cluster miRNAs can help us better understand how the alteration of the cluster miRNAs might contribute to development of cancers and shed light on the molecular mechanisms of miRNA function.

A. Benefits:

Signosis' miRNA plate arrays offer following benefits:

• Simple - Do miRNA detection as doing ELISA. It does not need enzymatic conversion from miRNA into cDNA. The entire assay procedure is “incubation and wash”.

• Higher sensitivity and better discrimination power - The detection is approximately 100 times more sensitive than miRNA Northern blot. In addition, it has higher discrimination power than miRNA Northern blot in differetiating miRNA isoforms.

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

B. Principle of the technology

In the proprietary miRNA plate assay, one miRNA molecule is flanked by a capture oligo and a biotinated detection oligo through two bridge oligos. One of the bridge oligos is partially hybridized with the miRNA molecule and the capture oligo and another one with the miRNA and the detection oligo. The hybrid is captured onto plate through hybridization with an immobilized oligo and detected by a streptavidin-HRP conjugate and chemiluminecscent substrate. This hybrid structure is sensitive to the sequence of the miRNA molecule. One nucleotide difference can prevent the formation of the hybrid and therefore miRNA isoform can be differentiated, which normally is hard to do with Northern blot. In addition, the sensitivity of the assay is higher than miRNA Northern blot assay.