Stem cell miRNA Array

Stem cells can differentiate into all of the specialized embryonic tissues during embryo development. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing specialized cells, in order to maintain the normal turnover of regenerative organs, such as blood, skin or intestinal tissues. Recent studies suggest that maintenance and differentiation of stem cells are regulated by microRNAs (miRNA). MiRNAs are small single-stranded RNA molecules (~20nt long), playing an important role in gene regulation and cell differentiation. MiRNAs are found to be differentially expressed in stem cells, such as brain-specific miR-124a and miR-9 molecules during neural lineage differentiation. In addition, miRNAs are also reported to regulate cancer stem cells. Signosis has developed a miRNA array specifically to target 69 miRNAs that are shown in literature to be associated with stem cell differentiation and maintenance. The array can be used for human, mouse and rat samples. Profiling the expression of these miRNAs will help to reveal the functions of miRNAs in stem cells.

A. Benefits:

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

• High discriminative power - 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 69 stem cell-associated 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.