Apoptosis-Associated miRNA Plate Array

Apoptosis involves a series of biochemical events leading to a characteristic cell morphology and death, including changes to the cell membrane, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. It plays important roles in removing unwanted cells during development and homeostasis. Excessive apoptosis causes hypotrophy, such as in ischemic damage, whereas an insufficient amount results in uncontrolled cell proliferation, such as cancer. MicroRNAs are newly discovered small non-coding RNA molecules that regulate up to 30% of mammalian gene expression. Numerous miRNAs have been revealed to have an influence on the regulation of apoptotic cell signaling such as overexpression of miR-145, miR-216, miR-182, and miR-96 miRNA to decrease caspase-3 activation. Signosis developed a miRNA array for targeting 47 miRNAs that have been shown in literature to be involved in apoptosis. Profiling the expression of these miRNAs will facilitate in uncovering the regulation of miRNA in apoptosis.

A. Benefits:

Signosis' apoptosis-associated miRNA plate array is a type of assays via direction hybridization of total RNA, which offers following benefits:

• Simple - Profiling the expression of 48 miRNA detection even simpler than 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

Signosis’ apoptosis-associated miRNA plate array is a simple two-step assay; plate hybridization and streptavidin-HRP detection. The plate is pre-coated with an oligo mix, including a pair of unique oligos that hybridize side-by-side to a specific target miRNA and a universal capture oligo and a biotin-labeled oligo. In the assay, total RNA is directly utilized for hybridization. When the target miRNA exists in RNA, it acts as a bridge to bring the biotin-labeled oligo to the capture oligo, which can be detected through streptavidin-HRP conjugate and a chemiluminescent substrate. If there exists no specific miRNA, the biotin-labeled probe will be washed away, leading to no detection. In the plate array, 48 wells are coated with different oligo mixes for different miRNAs. A single 96-well plate allows quantitative measurement and comparison of 48 miRNAs between two samples. U6 RNA is used for normalization.

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