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.

KEYWORDS:

• microRNA
• miRNA
• isomiR
• RT-qPCR
• biomarker
• NGS

Financial & competing interests disclosure

This work was supported in part by project grant ref. 1444 from Fight for Sight (London, UK).The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Key issues

• There are two main approaches to detecting microRNA: polyadenylation and oligo(dT) and microRNA-specific RT primer.

• Choice of RNA isolation method can influence the detection of microRNAs and should be optimized for the specific sample type under investigation. Direct lysis is an appealing alternative to simplify assays and reduce bias introduced during RNA extraction.

• MicroRNAs exist as a series of variants or isomiRs and the choice of RT-PCR strategy will determine whether all or only selected isomiRs will be detected. Polyadenylation followed by oligo(dT)-directed RT can detect all 3′ isomiRs whilst microRNA specific stem-loop RT focuses on one or a subset of isomiRs.

• Future techniques will likely combine RT-PCR with sequencing to provide quantitative data on all isomiRs present.

• Design of primers to discriminate between closely related microRNA sequences is challenging due to their short length, but can be aided by inclusion of high-affinity modified nucleotides.

• Careful primer design and use of hot start techniques to minimize amplification of off-target sequences and primer dimers maximizes the sensitivity and specificity of microRNA detection. New approaches are being developed to reduce propagation of primer dimers.

• Miniaturization of RT-PCR assays made possible with advances in liquid handling technology reduces reagent costs and facilitates evaluation of more microRNAs from limited samples.

• Appropriate normalization is critical to achieve reliable detection of microRNAs and a panel of endogenous controls empirically evaluated for the specific situation combined with exogenous ‘spike-ins’ is recommended.

• The availability of rapid thermal cycling devices is reducing the time required to detect microRNAs and it is now possible to complete the PCR stage in less than 10 minutes.