Quantification of mRNA using real-time RT-PCR
Abstract
The real-time reverse transcription polymerase chain reaction (RT-qPCR) addresses the evident requirement for quantitative data analysis in molecular medicine, biotechnology, microbiology and diagnostics and has become the method of choice for the quantification of mRNA. Although it is often described as a "gold" standard, it is far from being a standard assay. The significant problems caused by variability of RNA templates, assay designs and protocols, as well as inappropriate data normalization and inconsistent data analysis, are widely known but also widely disregarded. As a first step towards standardization, we describe a series of RT-qPCR protocols that illustrate the essential technical steps required to generate quantitative data that are reliable and reproducible. We would like to emphasize, however, that RT-qPCR data constitute only a snapshot of information regarding the quantity of a given transcript in a cell or tissue. Any assessment of the biological consequences of variable mRNA levels must include additional information regarding regulatory RNAs, protein levels and protein activity. The entire protocol described here, encompassing all stages from initial assay design to reliable qPCR data analysis, requires approximately 15 h.
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Use of a 3':5' integrity assay requires that the PCR efficiencies of both assays be virtually identical and not subject to differential inhibition.
“Use of a 3Ј:5Ј assay requires that the PCR efficiencies of both assays be virtually identical (51 ) and not be subject to differential inhibition.”
From The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments (2009) · cites this paper as [51]
Poor annealing optimization has a large effect on assay quality.
“poor annealing optimization has a large effect on assay quality (51 ).”
From The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments (2009) · cites this paper as [51]
Secondary structure disrupts primer binding and unfolding during extension.
“the fact that secondary structure disrupts primer binding and unfolding during extension is widely recognized (Peters et al. 2004; Nolan et al. 2006).”
From The paradox of dual roles in the RNA world: resolving the conflict between stable folding and templating ability (2013) · cites this paper as Nolan et al. 2006
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- Authors
- Nolan, T., Hands, R. E., & Bustin, S. A.
- Affiliations
- University of London (GB)
- Corresponding author
- Bustin, Stephen A
- Venue
- Nature Protocols
- Published
- 2006-08
- Volume
- 1
- Issue
- 3
- Pages
- 1559-1582
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- OpenAlex
- W2008183439
- Cited by
- 2,247
- References
- 61
- Citations by year
- 2026: 72 · 2025: 111 · 2024: 125 · 2023: 124 · 2022: 127 · 2021: 167 · 2020: 137 · 2019: 138 · 2018: 82 · 2017: 110 · 2016: 121 · 2015: 116 · 2014: 121 · 2013: 135 · 2012: 123
- FWCI
- 27.84
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene expression and cancer classification
- Keywords
- mRNA quantification, assay standardization, data normalization, assay design, reproducibility, mRNA levels
- MeSH terms
- Data Interpretation, Statistical, RNA, Messenger, Reverse Transcriptase Polymerase Chain Reaction, Gene Expression Profiling
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