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High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs
Published on: August 3, 2011
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Considerations for optimization of microRNA PCR assays for molecular diagnosis
Margaret Dellett1, David Arthur Simpson1
1a Centre for Experimental Medicine , Queen's University Belfast , Belfast , Northern Ireland , UK.
Expert Review of Molecular Diagnostics
|February 9, 2016
Summary
Measuring microRNAs and their variants (isomiRs) in biofluids using RT-qPCR presents challenges. This study details methods to improve detection sensitivity and specificity for these potential biomarkers.
Area of Science:
- Biomarker Discovery
- Molecular Biology
- Genomics
Background:
- MicroRNAs (miRNAs) are stable in biofluids, showing promise as biomarkers.
- Their small size and heterogeneity (isomiRs) complicate detection via RT-qPCR.
- Accurate quantification of miRNAs and isomiRs is crucial for biomarker applications.
Purpose of the Study:
- To outline key considerations for using RT-qPCR to measure plasma miRNAs and isomiRs.
- To discuss strategies for enhancing assay specificity and sensitivity.
- To explore novel approaches for miRNA quantification and characterization.
Main Methods:
- Incorporation of modified nucleotides into primer sequences for improved RT-qPCR performance.
- PolyA tailing with oligo(dT)-based reverse transcription for broad isomiR detection.
- Use of stem-loop RT oligonucleotides and sequence-specific probes for targeted isomiR analysis.
- Next-generation sequencing (NGS) of RT-PCR products for comprehensive quantification and characterization.
Main Results:
- Modified nucleotides enhance primer affinity, specificity, and sensitivity in RT-qPCR assays.
- PolyA tailing methods effectively capture a wide range of isomiRs.
- Specific RT strategies and probes allow for the detection of individual isomiRs.
- NGS provides a powerful tool for both quantifying and characterizing miRNA RT-PCR products.
Conclusions:
- Optimized RT-qPCR protocols, including modified nucleotides and specific RT strategies, can overcome challenges in miRNA and isomiR detection.
- Next-generation sequencing offers a comprehensive approach for analyzing miRNA expression profiles and isomiR heterogeneity.
- These advancements facilitate the reliable use of miRNAs as biomarkers in biofluid samples.
