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Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs
Published on: April 14, 2015
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A systemic approach to screening high-throughput RT-qPCR data for a suitable set of reference circulating miRNAs
Konrad Pagacz1,2, Przemyslaw Kucharski1,3, Urszula Smyczynska1
1Department of Biostatistics and Translational Medicine, Medical University of Lodz, Lodz, Poland.
BMC Genomics
|February 2, 2020
Summary
Finding a reliable reference for serum microRNA (miRNA) expression studies is challenging. This study identified a stable combination of three endogenous miRNAs for accurate miRNA profiling in serum samples.
Area of Science:
- Biochemistry
- Molecular Biology
- Genomics
Background:
- Lack of consensus on reference genes for serum/plasma miRNA expression quantitative PCR (qPCR) studies.
- Previous reference candidates for miRNA profiling lack convincing validation.
- Need for robust endogenous reference miRNAs in circulating cell-free body fluids.
Purpose of the Study:
- To develop and validate a new in silico approach for identifying suitable endogenous reference miRNAs in human serum.
- To establish a reliable reference combination for miRNA expression profiling.
- To improve the accuracy and reproducibility of serum miRNA quantification.
Main Methods:
- Utilized miRNA profiling data from Gene Expression Omnibus (GEO) for in silico analysis.
- Applied three normalization algorithms (NormFinder, BestKeeper, GeNorm) to identify stable miRNAs.
- Validated the identified reference miRNA set on two independent datasets.
Main Results:
- Discovered and validated a set of 13 candidate endogenous reference miRNAs.
- Demonstrated that combinations of 2 or 3 miRNAs provide more stable normalization than single miRNAs.
- Identified the arithmetic mean of miR-24, miR-222, and miR-27a as the most stable 3-miRNA reference combination.
Conclusions:
- No single miRNA is universally suitable as a reference for serum miRNA qPCR profiling.
- A carefully selected combination of endogenous miRNAs can provide stable and reliable normalization.
- The validated miRNA set offers a robust solution for accurate serum miRNA expression analysis.
