Real-time RT-PCR: considerations for efficient and sensitive assay design
Abstract
Real-time RT-PCR has been recognised as an accurate and sensitive method of quantifying mRNA transcripts. Absence of post amplification procedures allows rapid analysis with a greater sample throughput, yet with less risk of amplicon carry-over as reaction tubes are not opened. In order to maximise sensitivity, careful reaction design and optimisation is essential. Several aspects of assay design for real-time RT-PCR are discussed in this paper. We demonstrate the effect of amplicon secondary structure on reaction efficiency and its importance for primer design. Taq-man probes with a deoxyguanosine base at the 5' end fluoresce weakly when labelled with FAM, although weak fluorescence is not a problem when probes are labelled with Texas Red. DNA contamination of RNA samples purified using silica membrane columns is a significant problem but DNase digestion can be used to reduce this, particularly in-solution. MMLV and AMV enzyme systems using a variety of RT priming methods are compared and the problem of primer-dimer formation associated with RT enzymes is described.
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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 Peters et al. 2004
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- Authors
- Peters, I. R., Helps, C. R., Hall, E. J., & Day, M. J.
- Affiliations
- University of Bristol (GB)
- Corresponding author
- Peters, I.R
- ORCID
- Venue
- Journal of Immunological Methods
- Publisher
- Elsevier BV
- Published
- 2004-03
- Volume
- 286
- Issue
- 1-2
- Pages
- 203-217
- ISSN
- 0022-1759 (ISSN-L)
- OpenAlex
- W2122556781
- Cited by
- 159
- References
- 23
- Citations by year
- 2026: 1 · 2025: 3 · 2024: 1 · 2022: 2 · 2021: 3 · 2020: 6 · 2019: 6 · 2018: 2 · 2017: 4 · 2016: 5 · 2015: 6 · 2014: 7 · 2013: 14 · 2012: 5
- FWCI
- 4.39
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Journal of Immunological Methods (publishedVersion)
- PubMed (publishedVersion)
- Bristol Research (University of Bristol) (submittedVersion)
- Funders
- Waltham Centre for Pet Nutrition
- 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 › DNA and Nucleic Acid Chemistry
- Keywords
- real-time RT-PCR, assay design, primer design, TaqMan probes, DNase treatment, AMV reverse transcriptase, reaction efficiency, mRNA quantification
- MeSH terms
- Animals, Biopsy, Deoxyguanosine, Deoxyribonucleases, Dogs, Fluorescent Dyes, Glucosephosphate Dehydrogenase, Intestinal Mucosa, Nucleic Acid Conformation, RNA, Reverse Transcriptase Polymerase Chain Reaction
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