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Identification of extremely GC-rich micro RNAs for RT-qPCR data normalization in human plasma.
Volker Baumann1, Angelos-Theodoros Athanasiou1, Omid R Faridani2,3
1Genomics Core Facility, VetCore, University of Veterinary Medicine, Vienna, Austria.
Frontiers in Genetics
|January 23, 2023
Summary
This study identifies highly stable microRNA (miRNA) normalizers for human plasma reverse transcription-quantitative PCR (RT-qPCR), including novel, extremely guanine-cytosine-rich candidates. These findings improve miRNA quantification accuracy for potential diagnostic biomarkers.
Area of Science:
- Biochemistry and Molecular Biology
- Genomics and Genetics
- Biotechnology
Background:
- Accurate quantification of microRNAs (miRNAs) in human plasma is crucial for developing diagnostic biomarkers.
- Existing normalizers for reverse transcription-quantitative PCR (RT-qPCR) may not adequately represent the diverse miRNome, particularly guanine-cytosine (GC)-rich regions.
- Small-RNA sequencing (sRNA-seq) has limitations in detecting highly GC-rich miRNAs.
Purpose of the Study:
- To identify and validate high-quality miRNA normalizers for human plasma RT-qPCR, with a focus on extremely GC-rich sequences.
- To expand the repertoire of reliable normalizers for accurate miRNA expression analysis.
- To enhance the potential of plasma miRNAs as biomarkers for clinical phenotypes and stress reactions.
Main Methods:
- High-throughput microarray analysis was used to screen for stable miRNA candidates in plasma.
- Nine normalization approaches were applied to rank miRNA abundances based on consistency and uniformity.
- Stem-loop RT-qPCR was employed to validate the stability of selected normalizers in a cohort of 32 healthy individuals.
Main Results:
- Microarray analysis identified 11 stable miRNA sequences, including those with moderate (45%-65% GC) and extreme (80%-95% GC) content.
- Extremely GC-rich miRNAs, underrepresented in sRNA-seq, were found to be stable normalizers.
- RT-qPCR validation confirmed low inter-individual variance (0.6%-8.2% CV) for selected normalizers, with miR-3665 and miR-1915-3p showing superior stability.
- The best-performing duo normalization factor included an extremely GC-rich miRNA.
Conclusions:
- The identification of extremely GC-rich plasma normalizers significantly enhances the accuracy of PCR-based miRNA quantification.
- These novel normalizers improve the reliability of plasma miRNA analysis, supporting their use as biomarkers for early diagnosis and monitoring of clinical conditions.
- The validated miRNAs may also find applications in related animal models due to their conserved nature.
