Purelink pro 96 viral rna dna kit

Parallel cultures of MDCK cells were infected with influenza virus at an MOI of 0.1 in 24-well plates. At set time points after infection, the supernatants (extracellular viral genomes) or cells (intracellular viral genomes) from single wells were frozen. RNA was harvested from supernatants using Purelink Pro 96 viral RNA/DNA kit (12280; Invitrogen) and from cells using a Purelink Pro 96 RNA kit (12173011A). The number of M genome segments was measured by quantitative RT-PCR as described below (see “Specific infectivity assay”).

RNA was extracted from the supernatants of virally infected cells using either TRIzol reagent (15596; Life Technologies, Inc.) or a Purelink Pro 96 viral RNA/DNA kit (12280; Invitrogen). SuperScript III (18080; Invitrogen) was used to synthesize cDNA using random hexamers. Quantitative PCR was performed on a 7500 Fast real-time PCR system (Applied Biosystems). SuperScript III RT/Platinum Taq (Thermo 2574030) was used with the primers 5′-GACCRATCCTGTCACCTCTGAC-3′ and 5′-AGGGCATTYTGGACAAAKCGTCTA-3′, and the TaqMan probe 6-carboxyfluorescein (FAM)-TGCAGTCCTCGCTCACTGGGCACG-3′ with Blackhole Quencher 1 with an annealing temperature of 55°C for M segment copy number measurement. Quantification of cDNA copy number based on threshold cycle (C_T) values was performed using standard curves from 10-fold dilutions of plasmid containing the M gene of A/Puerto Rico/8/1934 H1N1. The ratio of the infectious titer per milliliter to the genome copy number per milliliter is the specific infectivity of the sample.