Figure 2

Quantitation of PB2, NA and NS vRNA (A) and mRNA (B) in cells infected with the wild-type (WT) or NSNA virus at different postinfection time points. Uni-12 primer (0.2 ng/μl) [35] was used for the cDNA synthesis of vRNA, whereas oligo dT₂₀ (25 μM) was used to generated cDNA of viral mRNA. In a typical reverse transcription reaction, 0.5 μg of DNase-treated RNA sample was mixed with 1 μl of the corresponding primer, 4 μl of 5x first stand buffer, 2 μl of 0.1M dithiothreitol, and 1 μl of 10 mM deoxyribonucleoside triphosphates (Strategene), 150 U of SuperScript II reverse transcriptase in a 20 μl reaction. For detecting NA and NS RNA species, RNase-treated cDNA was examined by 5'-nuclease-based assays in a 7300 Sequence Detection System (Applied Biosystems). Briefly, 5 μl of the corresponding diluted cDNA samples were mixed with 12.5 μl superMix-UDG (Invitrogen), 0.5 μl of Rox reference dye, 1 μl of 10 mM forward primer, 1 μl of 10 mM reverse primers, 1 μl of 10 mM probe and 4 μl of water. Reactions were first incubated at 50°C for 2 min, followed by 95°C for 10 min. Reactions were then thermal-cycled for 45 cycles (95°C for 15 sec, 56°C for 1 min). Primers used in the NA detection assay were 5'-ACCGACCATGGGTGTCCTT-3' (corresponds to nt 870–888 of the NA cRNA) and 5'-GAAAATCCCTTTACTCCGTTTGC-3' (complementary to nt 998–1020 of the NA cRNA). Primer used in the NS detection assay were 5'-TACCTGCATCGCGCTACCTA-3' (corresponds to nt 277–296 of the NS cRNA) and 5'-ATGATCGCCTGGTCCATTCT-3' (complementary to nt 378–397 of the NS cRNA) were used. The probes used in the NA and NS assays were 5'-FAM-CGTCCCAAAGATGGA-NFQ-3' (corresponds to nt 950–964 of the NA cRNA; FAM, 6-carboxyfluorescein; NFQ, nonfluorescent quencher) and 5'-VIC-CACTGGTTCATGCTCA-NFQ-3' (corresponds to nt 327–342 of the NA cRNA; VIC, a proprietary dye), respectively. For the quantitation of PB2 RNA species, cDNA samples were amplified by using FastStart DNA Master SYBR Green I kit (Roche) in a LightCycler platform (Roche). In a typical reaction, 5 μl of RNase-treated cDNA was mixed with 2 μl master mixtures, 1.6 μl of MgCl₂, 1 μl of forward primer (5'-CCGCAGTTCTGAGAGGATTC-3', corresponds to nt 2090–2109 of PB2 cRNA), 1 μl of reverse primer (5'-TCCGTTTCCGTTTCATTACC-3', complementary to nt 2226–2245 of the PB2 cRNA) and 1.6 μl of water. Reactions were first incubated at 95°C for 10 min, followed by a thermal-cycling (95°C for 10 sec, 58°C for 5 sec, 72°C for 15 sec; 40 cycles). The specificities of the amplified products were all confirmed by melting curve analysis. In all the PCR assays, serially diluted plasmids containing the corresponding sequences were used as standard controls. All the data were derived from three independent assays. The levels of mRNA and vRNA from the studied mutants were analyzed by two-tails paired t-test.