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.
Peters
I R
IR
School of Clinical Veterinary Science, University of Bristol, Langford House, Langford, Bristol BS40 5DU, UK. I.R.Peters@Bristol.ac.uk
Helps
C R
CR
Hall
E J
EJ
Day
M J
MJ
D000818:Animals; D001706:Biopsy; D003849:Deoxyguanosine / Q000009:adverse effects / Q000031:analogs & derivatives*; D003851:Deoxyribonucleases / Q000378:metabolism; D004285:Dogs; D005456:Fluorescent Dyes / Q000737:chemistry*; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics / Q000276:immunology; D007413:Intestinal Mucosa / Q000201:enzymology / Q000276:immunology; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards
D005456:Fluorescent Dyes; C070539:5'-deoxyguanosine; D012313:RNA; D005954:Glucosephosphate Dehydrogenase; D003851:Deoxyribonucleases; D003849:Deoxyguanosine