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

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