Let7 family is known to regulate proliferation and differentiation processes in species ranging from C- elegans to human. The mature forms (designated let-7a through let-7i ) differ at one to four positions from the let-7a sequence. The expression level of the isoforms is variable, which may play a diverse role in organ development and disease etiology. To distinguish these isoforms will help to elicit the mechanism of Let7 function. Based on its proprietary technology, Signosis has developed a highly sensitive and discriminative real-time PCR assay method for measuring miRNA expression of let7 isoforms (Let7a, Let7b, Let7c, Let7d, Let7e, Let7f, Let7g and Let7i) simultaneously. The assay implements oligo-ligation and SYBR green based real-time PCR, which can be used for quantitative analysis of miRNA expression in either total RNA or cell lysate without cDNA conversion.
In the assay, the target miRNA molecule is hybridized with two oligos 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. A single nucleotide difference among miRNAs will block either the hybridization or the ligation. After the pair of oligos is ligated, the ligated molecules are subjected to real-time PCR analysis.