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

IR Peters, CR Helps, EJ Hall, MJ Day

Research output: Contribution to journalArticle (Academic Journal)peer-review

116 Citations (Scopus)

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. Author Keywords: Author Keywords: Canine; Real-time RT-PCR; Primer–dimers; Reverse transcription; Genomic contamination; Taq-man probes AMV-RT, Avian myeloblastosis virus reverse transcriptase; gDNA, Genomic DNA; MMLV-RT, Moloney murine leukaemia virus reverse transcriptase; PCR, Polymerase chain reaction; RT, Reverse transcriptase; RT-PCR, Reverse transcriptase polymerase chain reaction
Translated title of the contributionReal-time RT-PCR: considerations for efficient and sensitive assay design
Original languageEnglish
Pages (from-to)203 - 217
Number of pages15
JournalJournal of Immunological Methods
Volume286 (1-2)
DOIs
Publication statusPublished - Mar 2004

Bibliographical note

Publisher: Elsevier

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