title: Considerations for optimization of microRNA PCR assays for molecular diagnosis.
abstract: The remarkable stability of microRNAs in biofluids underlies their potential as biomarkers, but their small size presents challenges for detection by RT-qPCR. The heterogeneity of microRNAs, with each one comprising a series of variants or 'isomiRs', adds additional complexity. Presented here are the key considerations for use of RT-qPCR to measure microRNAs and their isomiRs, with a focus on plasma. Modified nucleotides can be incorporated into primer sequences to enhance affinity and provide increased specificity and sensitivity for RT-qPCR assays. Approaches based upon polyA tailing and use of a common oligo(dT)-based reverse transcription oligonucleotide will detect most isomiRs. Conversely, stem-loop RT oligonucleotides and sequence specific probes can enable detection of specific isomiRs of interest. Next generation sequencing of all the products of a microRNA RT-PCR reaction is a promising new approach for both microRNA quantification and characterization.
authors: a Centre for Experimental Medicine , Queen's University Belfast , Belfast , Northern Ireland , UK.
authors: Margaret
authors: M
authors: Dellett
authors: a Centre for Experimental Medicine , Queen's University Belfast , Belfast , Northern Ireland , UK.
authors: David Arthur
authors: DA
authors: Simpson
mesh_terms: D015415:Biomarkers / Q000097:blood / Q000737:chemistry; D006801:Humans; D035683:MicroRNAs / Q000097:blood* / Q000737:chemistry; D025202:Molecular Diagnostic Techniques / Q000379:methods* / Q000592:standards; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D017423:Sequence Analysis, RNA / Q000379:methods
chemical_list: D015415:Biomarkers; D035683:MicroRNAs
keywords: NGS; RT-qPCR; biomarker; isomiR; miRNA; microRNA