Patent-pending miRNA Two-Tailed RT-qPCR technology shows exceeding performance compared to other techniques.
The challenge detecting small microRNAs is that two conventional PCR primers do not fit the target as their combined length is almost twice the length of the microRNA. Older techniques have solved this by extending the microRNA using e.g., a hairpin primer, adding a poly A-tail, or adding a fragment by ligation. This, however, compromises the assay sensitivity and specificity, as only one of the PCR primers sense the actual microRNA sequence; the other senses the added extension. Further, these methods fail to detect microRNAs modified in the 3’-end as it interferes with the extension process.
The technology has been developed in TATAABiocenter and bought by BioVendor. The miRNA two-Tailed RT-qPCR assays offers a superior solution. Instead of using a single binding probe, Two-tailed PCR uses two hemiprobes, which bind to different stretches of the microRNA, that are connected by a folded tether. While each hemiprobe is too short to bind the microRNA, when both hemiprobes are complementary they bind cooperatively. Binding is exceeding specific, as a mismatch is much more profound in a short hemiprobe. The cDNA formed can then be PCR amplified using two sequence specific primers. SYBR used for detection. High melting resolution analysis can be used for non-specific products detection.