Cancer miRNA array

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate up to 30% of mammalian gene expression. So far, more than 400 miRNAs have been identified in the human genome and many of them are only different in one or a few nucleotides. Expression of mature miRNAs is tissue-specific and the abundancy of miRNAs varies in several orders of magnitude. Aberrant expression of miRNAs may be involved in human cancers. Systematic profiling of miRNA expression displays unique signatures in a number of cancers. Profiling comparison of miRNA expression displays a set of miRNAs differentiated in cancers. Signosis cancer miRNA array focuses on profiling these cancer-related miRNAs.

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 the 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, differing by 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 hybridize with the target miRNA to form a RNA/DNA duplex. When the sequences are perfectly matched, they are aligned with the miRNA and the joint can be ligated with 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 miRNA, the hybrid might not be stable; therefore we introduce the stacking sequences. By extending these two oligos 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 the array hybridization.

We offer miRNA profiling assay kits to profiling the expression of 132 of the most popular miRNAs and their isoforms. The procedure is simple and straight forward, including 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 strand; and (3) amplify the ligated DNA with T7 transcription.