Product Name Catalog # Price   Qty
Apoptosis-Associated miRNA Array AP-0004 $469
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Characterization

Description:

Numerous miRNAs have been reported to have an influence on the regulation of apoptotic cell signaling such as the overexpression of miR-145, miR-216, miR-182, and miR-96 miRNA to decrease caspase-3 activation. Signosis has developed a miRNA array for targeting 52 miRNAs that have been shown in literature to be involved in apoptosis.

 


Applicable Grid:

List of Applicable miRNAs

Let-7a-5p Let-7b-5p Let-7c-5p Let-7d-5p Let-7e-5p Let-7f-5p Let-7g-5p Let-7i-5p miR-1 miR-7-5p
miR-10a-5p miR-15a-5p miR-15b-5p miR-16-5p miR-21-5p miR-25-3p miR-28-5p miR-133b miR-145-5p miR-148
miR-155-5p miR-182-5p miR-204-5p miR-216-5p miR-224-5p mir-376c-3p miR-24-3p miR-101 miR-142 miR-150-5p
miR-151-3p miR-153-3p miR-188-5p miR-196a-5p miR-197-3p miR-202-5p miR-210-3p miR-214-3p miR-218-5p miR-296-5p
miR-144 miR-152 miR-184 miR-337 miR-338 miR-345 miR-371 miR-96 miR-369-5p miR-369-3p
miR-193a miR-193b RUN48              

Principle

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, only differing by 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 between isoforms, or are not sensitive enough to monitor low abundant miRNAs.

In our array assay, each miRNA molecule is targeted by two oligos that hybridize with the target miRNA to form a RNA/DNA duplex. When the sequences are perfectly matched, the oligos 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; Thus, miRNA isoforms can be differentiated. Due to the small size of miRNA, the hybrid might not be stable; Therefore, 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. In this way, each isoform can be easily distinguished by array hybridization.

The assay procedure includes three steps: (1) mix the miRNA with 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.


Data



Figure 1. Apoptosis miRNA analysis.  
5µg of total RNAs from U973, a p53 negative cell line, and A431, a p53 positive cell line, were assayed with human apoptosis miRNA array and detected with a chemiluminescence imaging system. The internal control RNU48 was indicated in the image.

 

Literature

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