Magna pure lc machine

Twenty-four hours after inoculation with rNDV at m.o.i. 3, cells were lysed with 300 µL lysis buffer of the Total Nucleic Acid Isolation kit (Roche) and RNA was isolated using a MagNA Pure LC machine (Roche) following the manufacturer’s instructions. qRT-PCR (30 cycles) was performed with 20 μL RNA in an ABI PRISM 7000 Sequence Detection System (Life Technologies), using TaqMan gene expression assay for human IFNβ1 (Hs00277188_s1, Life Technologies). The primers in this assay map to the extreme 3′ end of the hIFNβ gene (Hs00277188_s1, www.lifetechnologies.com) [29 ], with the reverse primer annealing downstream of the stop codon of the hIFNβ coding sequence. This region is not present in the rNDV-hIFNβ-F₀ virus. Therefore, the qRT-PCR assay is not able to detect IFN-mRNA transcribed from the virus, and the assay will only detect endogenous transcribed IFN-mRNA. To detect both endogenous and exogenous expressed IFN, primers and probes mapping in the IFN coding region were used with an in-house developed assay and β-actin was used as household gene). The sequences of the primers and probes for the IFN-coding region and β-actin have been described before [30 (link)]. Results are presented as fold change of inoculated samples versus mock-inoculated samples (duplicates), calculated using the 2^−ΔΔC_T method [31 (link)].

BAL samples stored in TRIzol were processed according to the manufacturer’s instructions to isolate RNA. Tissue samples stored in RNAlater were weighed, thawed, transferred to tubes containing a quarter-inch-diameter ceramic sphere in virus transport medium, and homogenized using a FastPrep 24 tissue homogenizer (MP Biomedicals, Eindhoven, The Netherlands). The homogenates were centrifuged, and the cleared supernatants were used for RNA isolation. 200 µL of the cleared supernatant of tissue samples, as well as 200 µL of other samples (transport medium of swabs, plasma) were combined with 300 µL lysis buffer of the Total Nucleic Acid Isolation kit (Roche, Woerden, The Netherlands) and RNA was isolated in a volume of 50 µL using a MagNA Pure LC machine (Roche) following the manufacturer’s instructions.
NDV-specific qRT-PCR was performed on 5 µL (TRIzol samples) or 19.5 µL (MagNA Pure samples) RNA in an ABI PRISM 7000 Sequence Detection System using TaqMan Fast Virus 1-Step Master Mix (both from Life Technologies) in a total volume of 30 µL, using primers as described by Wise et al. [21 (link)]. The RT step was 5 min at 50 °C, followed by 95 °C for 20 s. Cycling consisted of 45 cycles of 3 s denaturation at 95 °C, 5 s annealing at 54 °C and 31 s extension at 60 °C.