Method to measure precursor and mature miRNA specifically.
- Unique primers can specifically identify miRNAs and precursor miRNAs by standard qRT-PCR without the requirement for an additional detection probe.
- Allows for amplification and quantification of both mature miRNA and precursor miRNAs.
- Potential technology to evaluate disease-specific biomarkers.
MicroRNAs (miRNAs) are highly processed small RNAs that regulate protein expression. The miRNA expression levels have been linked to several disease states, such as cancer, Alzheimer’s and Parkinson’s disease. In order to accurately assess miRNA levels in relation to a disease state, it is critical to distinguish between immature and mature forms of miRNA but, the size of mature miRNAs makes detection difficult, as they are too small to detect via standard quantitation techniques. Current identification requires a lengthy process that includes preliminary isolation from whole tissues followed by a secondary enrichment for small RNAs. Despite these additional processing steps, a mature miRNA still cannot be distinguished from its pre-processed immature form using standard procedures. Therefore, there is a need to develop a method to accurately detect and quantify mature miRNAs for diagnosis of disease states or research purposes.
Emory researchers developed a technology to identify and distinguish mature miRNAs from precursor mRNAs after isolation and enrichment from tissues. The inventors designed unique short single-stranded nucleic acid sequences (primers) that amplify only the mature miRNA from an RNA mixture. This new primer design strategy can be used to quantitatively distinguish between pre-miRNAs and mature miRNAs via standard qRT-PCR. These primers were successfully used to detect the mature miRNAs: miR-16-1, miR-21-3P, and miR-132-3P associated with some types of cancer such as glioblastoma.
This technology has been successfully tested to detect 1) miRNAs associated with cancer during in vitro experiments, 2) both miRNAs and precursor miRNAs associated with PML knockout conditions, and 3) altered miRNAs and precursor miRNAs under Alzheimer’s patient brains and Alzheimer-like animal models.