Expression profiling of microRNA using real-time quantitative PCR, how to use it and what is available
Abstract
We review different methodologies to estimate the expression levels of microRNAs (miRNAs) using real-time quantitative PCR (qPCR). As miRNA analysis is a fast changing research field, we have introduced novel technological approaches and compared them to existing qPCR profiling methodologies. qPCR also remains the method of choice for validating results obtained from whole-genome screening (e.g. with microarray). In contrast to presenting a stepwise description of different platforms, we discuss expression profiling of mature miRNAs by qPCR in four sequential sections: (1) cDNA synthesis; (2) primer design; (3) detection of amplified products; and (4) data normalization. We address technical challenges associated with each of these and outline possible solutions.
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miR-33 shows tissue-dependent differences in 3' end length (isomirs) that might confound quantification by reverse transcription using hairpin primers.
“In these studies, we are relying on Northern blot analysis, because miR-33 shows tissue-dependent differences in 3Ј end length (Isomirs), which might confound quantification of miRNA lev- els by methods that rely on reverse transcription using hairpin primers (supplemental Fig. S13) (37,38).”
From Expression of miR-33 from an SREBP2 Intron Inhibits Cholesterol Export and Fatty Acid Oxidation* (2010) · cites this paper as [37]
Cites
Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method
Livak, K. J., & Schmittgen, T. D. (2001) · Methods · doi:10.1006/meth.2001.1262 · Cited by 180,860 · see citing works
A microRNA array reveals extensive regulation of microRNAs during brain development
KRICHEVSKY, A. M., KING, K. S., DONAHUE, C. P., KHRAPKO, K., & KOSIK, K. S. (2003) · RNA · doi:10.1261/rna.5980303 · Cited by 1,040 · see citing works
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Expression of miR-33 from an SREBP2 Intron Inhibits Cholesterol Export and Fatty Acid Oxidation*
Gerin, I., Clerbaux, L.-A., Haumont, O., Lanthier, N., Das, A. K., Burant, C. F., Leclercq, I. A., MacDougald, O. A., & Bommer, G. T. (2010) · Journal of Biological Chemistry · doi:10.1074/jbc.m110.152090 · Cited by 382 · see citing works
- Authors
- Benes, V., & Castoldi, M.
- Affiliations
- Heidelberg University (DE)
- Corresponding author
- Castoldi, Mirco
- ORCID
- Castoldi, Mirco
- Venue
- Methods
- Publisher
- Elsevier BV
- Published
- 2010-04
- Volume
- 50
- Issue
- 4
- Pages
- 244-249
- ISSN
- 1046-2023 (ISSN-L)
- OpenAlex
- W2169234352
- Cited by
- 361
- References
- 2
- Citations by year
- 2026: 4 · 2025: 8 · 2024: 17 · 2023: 9 · 2022: 17 · 2021: 20 · 2020: 24 · 2019: 33 · 2018: 25 · 2017: 27 · 2016: 20 · 2015: 31 · 2014: 34 · 2013: 32 · 2012: 31
- FWCI
- 2.58
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Methods (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Universität Heidelberg
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene expression and cancer classification
- Keywords
- miRNA expression profiling, real-time quantitative PCR, microRNA quantification, cDNA synthesis, primer design, data normalization, technical challenges
- MeSH terms
- Diamines, Fluorescent Dyes, Organic Chemicals, Quinolines, DNA, Complementary, Reverse Transcriptase Polymerase Chain Reaction, Oligonucleotide Array Sequence Analysis, Gene Expression Profiling, MicroRNAs, Benzothiazoles
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