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Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR
Published on: June 26, 2016
Sensitive PCR-based quantitation of cell-free circulating microRNAs
Michelle L Hastings1, Jaime Palma, Dominik M Duelli
1Department of Cell Biology and Anatomy, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA.
Methods (San Diego, Calif.)
|August 14, 2012
Summary
Cell-free microRNAs (miRNAs) in blood are valuable biomarkers. Standardized protocols for blood collection and miRNA analysis are crucial for accurate, reproducible quantification of these circulating RNA molecules.
Area of Science:
- Biochemistry
- Molecular Biology
- Biomarker Discovery
Background:
- Cell-free microRNAs (miRNAs) circulating in blood show promise as disease biomarkers.
- Quantifying circulating miRNAs using Taq-polymerase-based methods has identified correlations with various pathologies.
- Challenges exist in reproducible, quantitative assessment of circulating RNA due to blood's unique properties.
Purpose of the Study:
- To highlight critical factors affecting the accurate quantification of circulating cell-free miRNAs.
- To emphasize the need for standardized protocols in miRNA biomarker development.
- To guide researchers in achieving reproducible and sensitive miRNA analysis from plasma.
Main Methods:
- Review of factors influencing miRNA quantitation in plasma.
- Consideration of collection tube types, anticoagulants, and stabilizers.
- Analysis of sample processing, miRNA enrichment, RNA extraction, and enzyme selection.
Main Results:
- Blood components can interfere with RNA amplification and analysis enzymes.
- Inconsistent methodologies lead to challenges in reproducible miRNA quantitation.
- Several pre-analytical and analytical variables significantly impact miRNA measurement accuracy.
Conclusions:
- Standardized protocols are essential for reliable use of circulating miRNAs as biomarkers.
- Careful consideration of sample handling and analysis steps is necessary for accurate quantitation.
- Addressing these factors will enhance the utility of miRNAs in clinical diagnostics.
