Author: 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.
Title: Recurrent rhinovirus infections in a child with inherited MDA5 deficiency Document date: 2017_7_3
ID: vipx6t7e_67
Snippet: At 6, 24, or 48 h of RSV-GFP infection, infected cell cultures were washed twice with PBS to remove nonadherent cells, and total RNA was isolated using TRIzol extraction (Invitrogen) according to manufacturer's instructions. 0.5 to 2.0 µg total RNA was reverse transcribed using High-Capacity cDNA Reverse Transcription kit with RNase Inhibitor (Applied Biosystems). Diluted cDNA (1:5 to 1:10 in H 2 O) was analyzed for RSV N gene transcripts by qua.....
Document: At 6, 24, or 48 h of RSV-GFP infection, infected cell cultures were washed twice with PBS to remove nonadherent cells, and total RNA was isolated using TRIzol extraction (Invitrogen) according to manufacturer's instructions. 0.5 to 2.0 µg total RNA was reverse transcribed using High-Capacity cDNA Reverse Transcription kit with RNase Inhibitor (Applied Biosystems). Diluted cDNA (1:5 to 1:10 in H 2 O) was analyzed for RSV N gene transcripts by quantitative real-time PCR using TaqMan Universal PCR Master Mix on a 7500 Real Time PCR System (Applied Biosystems) per the manufacturer's instructions. The N gene forward primer sequence was 5′-TGG CAT GTT ATT AAT CAC AGA AGA TGCT-3′, N gene reverse primer sequence was 5′-TTC TCT TCC TAA CCT AGA CAT CGC ATA-3′, and the N gene probe sequence was 5′-6FAM-AAC CCA GTG AAT TTA TG-MGB-NFQ-3′. Viral copy numbers were calculated based upon a standard curve generated from RSV-GFP virion RNA and were shown relative to the RSV transcript levels in the father's cells.
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