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Updated: May 23, 2026
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Quantitative Real-Time Polymerase Chain Reaction Evaluation of MicroRNA Expression in Kidney and Serum of Mice with Age-Dependent Renal Impairment
Published on: April 29, 2022
Comprehensive evaluation of reference miRNA for extracellular miRNA quantification in CKD
Anvesha Srivastava1, Mark Maienschein-Cline2, Ana Pabalan1
1Division of Renal Diseases and Hypertension, George Washington University, Washington DC, USA.
Methods (San Diego, Calif.)
|May 21, 2026
Summary
This study identified hsa-miR-24-3p as the most stable endogenous reference microRNA (miRNA) for normalizing extracellular miRNA quantification in plasma. Urinary miRNAs require further optimization for reliable normalization in chronic kidney disease research.
Area of Science:
- Biomolecular analysis
- Molecular diagnostics
- Biomarker discovery
Background:
- Extracellular microRNAs (miRNAs) are promising biomarkers and therapeutic targets.
- Reliable quantification of extracellular miRNAs using RT-qPCR necessitates appropriate normalization.
- A universal endogenous reference miRNA for extracellular analysis has not been established.
Purpose of the Study:
- To identify optimal endogenous control miRNAs for extracellular miRNA quantification.
- To assess miRNA stability in plasma and urine samples from chronic kidney disease patients.
Main Methods:
- Next-generation sequencing identified candidate reference miRNAs in plasma and urine.
- NormFinder algorithm and complementary stability metrics prioritized candidates.
- RT-qPCR validated top candidate miRNA stability in independent cohorts.
Main Results:
- Hsa-miR-24-3p demonstrated superior expression stability and detectability in plasma (97%), establishing it as a robust endogenous reference.
- Hsa-miR-126-3p and hsa-miR-30e-5p also showed high stability in plasma; dual normalization with hsa-miR-30e-5p improved stability.
- Urinary reference miRNAs exhibited low detectability and insufficient stability for routine normalization without optimization.
Conclusions:
- Hsa-miR-24-3p is a reliable endogenous control for plasma miRNA normalization in chronic kidney disease.
- Urinary miRNA quantification requires context-specific validation and technical optimization for accurate results.
- This study provides crucial guidance for accurate and reproducible extracellular miRNA analysis.
