miRNA Plate Assay

MicroRNAs (miRNAs) are small noncoding RNAs that control gene expression at the posttranscriptional level through selectively binding to complementary messenger RNA sequences. Approximately 30% of mammalian genes are regulated by these small RNA molecules, which lead to the regulation of various biological functions, including development, cell differentiation, proliferation, apoptosis, and maintenance of stemness and imprinting. Northern blotting has been the most commonly used method for analyzing individual miRNAs, although more recently a number of new approaches have been described, including the related real-time PCR and invader assays for quantifying individual miRNAs. Low sensitivity and poor throughput are the main issue of Northern blot analysis. Signosis’ proprietary miRNA plate assay does not need biochemical conversion of miRNA molecules into cDNAs, and the procedure of the assay is as simple as incubation and wash.

To analyze a miRNA with the plate assay, you need to purchase the common component miRNA plate assay kit (MA-0101) and a specific oligo mix (MO-XXXX) corresponding to the miRNA as listed in the figure 1.

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 a specific miRNA 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.