Human Apoptosis-Associated miRNA 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 52 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:

Based on our proprietary T7-OLA (oligo ligation assay) amplification technology, we are offering miRNA array kits with following benefits:

• High discriminative power - It is able to differentiate all miRNA isoforms. All let7 isoforms can be clearly distinguished in the assay.

• Linear amplification - Captured miRNAs are subjected to T7 amplification without introduction of bias in PCR.

• No pre-isolation of miRNA - Total RNA can be directly used for the assay without pre-isolation of miRNA.

• Simple procedure – The assay is simple and straightforward.

B. Principle of the technology

miRNA is different from large messenger RNA in three aspects; (1) miRNAs are small size molecules with quite a big difference in abundance, (2) mature miRNAs co-exist with their precursor pre-miRNA and pri-miRNA, which only differ in length, and (3) many miRNAs are very closely related in sequences, such as isoforms, with the difference of only one or a few nucleotides. Therefore, the conventional mircoarray technologies cannot directly be applied to analyzing these molecules. A number of miRNA microarray products are commercially available, but they are either tedious in requiring pre-isolation of microRNA, lack the discriminative power to differentiate isoforms, or are not sensitive enough to monitor low abundant miRNAs.

In our array assay, each miRNA molecule is targeted by two oligos that hybridizes with the target miRNA to form an RNA/DNA duplex. When the sequences are perfectly matched, these are aligned with the miRNA and the joint can be ligated by DNA ligase (figure 1). A single nucleotide difference among miRNAs will block either the hybridization or the ligation, so that miRNA isoforms can be differentiated. Due to the small size of miRNAl, the hybrid might not be stable; here we introduce the stacking sequences. By extending these two oligos along with their complementary oligos the stability is increased. Once the pair of oligos is ligated, the ligated molecules are subjected to linear amplification via T7 transcription into RNA in the presence of biotin-UTP, which are used as probes for array hybridization. To differentiate each isoform, we assigned unique tag sequences to the ligation oligos, so that single nucleotide differences are converted into unique tag sequences. Therefore, each isoform can be easily distinguished by array hybridization.

We offer miRNA profiling assay kits to profile the expression of 52 apoptosis-associated human miRNAs and their isoforms. The procedure is simple and straight forward, including three steps: (1) mix the miRNA with the provided oligos to form miRNA/oligo hybrids; (2) select the hybrids and remove free oligos, and ligate miRNA-directed pairing of oligos to become a single DNA; and (3) amplify the ligated DNA with T7 transcription.