Newly discovered microRNAs (miRNAs) are important to the regulation of gene expression, and up to 30% of mammalian genes may be regulated by miRNAs. 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. The expression of mature miRNAs is tissue-specific and the abundance of miRNAs varies over several orders of magnitude. More importantly, mis-regulation of miRNA expression may contribute to human cancers. Systematic profiling of miRNA expression plays the key role in the research of a number of cancers.
Signosis' Human miRNA array I allows you to profile 60 of the most popular miRNAs and their isoforms.
List of Applicable miRNAs
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.
miRNA analysis of miRNA expression in human brain and liver.
5µg RNA was incubated and annealed with DNA oligo mix. DNA-RNA hybrids were selected and ligated. The ligated molecules were amplified and labeled with T7 RNA polymerase in the presence of biotin-UTP. The labeled RNAs were hybridized with miRNA array, and detected with chemiluminescence imaging system.
LiteratureView user manual
MicroRNA-21 is Induced by Rapamycin in a Model of Tuberous Sclerosis (TSC) and Lymphangioleiomyomatosis(LAM). Trindade AJ, Medvetz DA, Neuman NA, Myachina F, Yu J, Priolo C, Henske EP. PLoS One. 2013;8(3):e60014.