Low copy number detection of herpes simplex virus type 1 mRNA and mouse Th1 type cytokine mRNAs by Light Cycler quantitative real-time PCR.
The real-time principle of reverse transcriptase polymerase chain reaction (RT-PCR) provides a quantitative and reproducible method to detect low copy number transcripts. The quantitative detection of cytokines from tissue samples is complicated by the low expression rates and the short half-lives of the cytokine proteins. The methods have been insensitive and labor-intensive. The LightCycler technique provides a 30-min PCR system with continuous fluorescent detection, analysis of the melting points of the products and user-friendly software for the analysis of the unknown samples. External copy number standards enable the measurement of amounts of the desired targets. We demonstrate the dynamic range of the RT-PCR system from a 100 to 10(7) mRNA copies of the mouse Th1 cytokines interleukin- (IL-) 12p35, 12p40 and IL-23p19 as well as gamma interferon (IFN-gamma) and the housekeeping gene beta-actin, with the usage of fluorescent hybridization probes. The cytokine quantitation was exemplified in murine nervous system samples. A viral transcript, mRNA of alpha trans-inducing factor (alphaTIF), or VP16 gene, of herpes simplex virus (HSV) type 1 was used to quantitate the viral replication in infected cells and in murine nervous system samples. For this viral transcript the linear dynamic range spanned from ten copies to one million copies (highest tested). For all tested cytokine transcripts, the detection level with the dsDNA binding dye SYBR Green I was one log lower than with the hybridizing fluorescent probes. The viral transcript was detected even with the SYBR Green I system at the level of ten copies. The specificity of the PCR was reached with the use of TaqStart antibody, by careful design of primers and probes, by melting temperature analysis and comparison with the gel electrophoresis and Southern blot analysis.
Broberg
Eeva K
EK
Department of Virology, University of Turku, Kiinamyllynkatu 13, FIN-20520, Turku, Finland. eeva.broberg@utu.fi
Nygårdas
Michaela
M
Salmi
Aimo A
AA
Hukkanen
Veijo
V
D000818:Animals; D002522:Chlorocebus aethiops; D016207:Cytokines / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis*; D005456:Fluorescent Dyes / Q000378:metabolism; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D006801:Humans; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics; D015203:Reproducibility of Results; D018417:Th1 Cells / Q000378:metabolism*; D014709:Vero Cells
D016207:Cytokines; D004279:DNA, Viral; D005456:Fluorescent Dyes; D012333:RNA, Messenger