Virus culture and virus detection by real-time PCR
Camelpox strain CP-19, CMV strain AD169, HHV-6 strain U1102, SARS coronavirus strain 6109 and YFV strain 17D were propagated according to standard procedures [8–10].
The respective MOI and time of cell culture are shown in table 1 and were chosen to allow maximal infection as determined by immunofluorescence and real-time PCR [8–11]. For kinetic studies, cells were harvested at several time points (table 1) and RNA was extracted. The RNA transcription level of putative reference genes was determined by quantitative real-time PCR as described below.
Extraction of RNA
Total RNA from 1 × 106 cells was prepared using the QIAamp RNA Blood Mini Kit and RNase-free DNase set (Qiagen, Hilden, Germany) according to the manufacturer's recommendations for cultured cells. RNA solution was treated with DNA-free (Ambion, Huntingdon, United Kingdom).
cDNA synthesis
cDNA was produced using the Superscript III RT-PCR System (Invitrogen, Karlsruhe, Germany) according to the manufacturer's recommendations for oligo(dT)20 primed cDNA-synthesis. cDNA synthesis was performed using 1 μg of RNA, at 50°C. Finally, cDNA was diluted 1:5 before use in QPCR.
Quantitative TaqMan PCR
Primers, TaqMan probes and QPCR conditions for reference gene analysis were used as previously described [5]. PCR was performed in a Perkin Elmer 7700 Sequence Detection System in 96-well microtiter plates using a final volume of 25 μl.
Calculations
Analysis was performed with the BestKeeper [6] and GeNorm [7] tools. The ΔΔCT value was calculated as follows: First the ΔCT for each time point of probe assessment between virus and Mock infected cells was calculated. In a second step the maximal differences between the time points were calculated as ΔΔCT.