Mouse lungs were isolated and snap-frozen in liquid nitrogen or suspended in Allprotect Tissue Reagent (Qiagen, Hilden,Germany). RNA was extracted as directed using the Qiagen RNeasy Mini Kit (Qiagen, Hilden,Germany). cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). qPCR was conducted using SsoAdvanced universal probes supermix (Bio-Rad, Hercules, CA) and target specific TaqMan real-time PCR assay primer probes (ThermoFisher Scientific, Waltham, MA). Viral burden was determined by quantitative real-time RT-PCR on lung RNA for viral matrix protein (M1) as described previously (38 (link), 39 (link)). Forward Primer:5′-GGACTGCAGCGTAGACGCTT-3′, Reverse Primer:5′-CATCCTGTTGTATATGAGGCCCAT-3′, Probe:5′-/56-FAM/CTCAGTTAT/ZEN/TCTGCTGGTGCACTTGCCA/3IABkFQ/−3′.
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Quantifying Viral Burden in Mouse Lungs
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Corresponding Organization : Children's Hospital of Pittsburgh
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Variable analysis
independent variables
- Isolation method (snap-frozen in liquid nitrogen or suspended in Allprotect Tissue Reagent)
- Viral burden (determined by quantitative real-time RT-PCR on lung RNA for viral matrix protein (M1))
- Mouse lungs
- RNA extraction using Qiagen RNeasy Mini Kit
- CDNA synthesis using iScript cDNA synthesis kit
- QPCR using SsoAdvanced universal probes supermix and TaqMan real-time PCR assay primer probes
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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