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PubMed · 15087233

Real-time RT-PCR: considerations for efficient and sensitive assay design.

I R Peters·C R Helps·E J Hall·M J Day
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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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AnimalsBiopsyDeoxyguanosineDeoxyribonucleasesDogsFluorescent DyesGlucosephosphate DehydrogenaseIntestinal MucosaNucleic Acid ConformationRNAReverse Transcriptase Polymerase Chain ReactionComparative StudyJournal ArticleResearch Support, Non-U.S. Gov't

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I R Peters, C R Helps, E J Hall, M J Day. 2004. Real-time RT-PCR: considerations for efficient and sensitive assay design.. https://doi.org/10.1016/j.jim.2004.01.003

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