Recurrent rhinovirus infections in a child with inherited MDA5 deficiency

Lamborn, Ian T.; Jing, Huie; Zhang, Yu; Drutman, Scott B.; Abbott, Jordan K.; Munir, Shirin; Bade, Sangeeta; Murdock, Heardley M.; Santos, Celia P.; Brock, Linda G.; Masutani, Evan; Fordjour, Emmanuel Y.; McElwee, Joshua J.; Hughes, Jason D.; Nichols, Dave P.; Belkadi, Aziz; Oler, Andrew J.; Happel, Corinne S.; Matthews, Helen F.; Abel, Laurent; Collins, Peter L.; Subbarao, Kanta; Gelfand, Erwin W.; Ciancanelli, Michael J.; Casanova, Jean-Laurent; Su, Helen C.

After washing off nonadherent cells twice with PBS, total RNA was isolated using TRIzol extraction (Invitrogen). 2 µg total RNA was reverse transcribed using High-Capacity cDNA Reverse Transcription kit with RNase Inhibitor (Applied Biosystems). Diluted cDNA was analyzed by quantitative real-time PCR using TaqMan Universal PCR Master Mix on a 7500 Real Time PCR System (Applied Biosystems), as previously described (Lee et al., 2015c) . The forward primer D110 was 5′-CTA GCC TGC GTGG-3′, reverse primer RVQ1 was 5′-AAA CAC GGA CAC CCA AAG TAGT-3′, and probe RVQ2 was 5′-6FAM-TCC TCC GGC CCC TGA-MGB-NFQ-3′. Viral copy numbers were calculated based upon a standard curve generated from HRV-B14 virion RNA and were shown relative to siRNA negative control. For infection of nasal epithelial cells, viral copy numbers were shown relative to values from father's cells.