Fig. 8

Gene knockdown and the effect on LGTV replication and production in IRE/CTVM19 cells. IRE/CTVM19 cells were treated with dsRNA to silence differentially-expressed transcripts and were subsequently infected with LGTV at an MOI of 0.5. a Transcripts coding for Argonaute (Ago 30) and Dicer (Dcr 90) were amplified by RT-PCR using dsT7-Ago 30 or dsT7-Dcr90 primers and visualised by agarose gel electrophoresis. A representative 1 % agarose gel from one of the three experiments is shown; upper lanes show Ago 30 and Dcr 90 PCR products, lower lanes show beta actin PCR products. b Gel-electrophoresis images were used to semi-quantify mRNA knockdown of Ago 30 and Dcr 90 with Image Lab software (BioRad) normalised to beta actin control. c Knockdown of mRNA was quantified using qRT-PCR. Gene expression was normalised to beta actin and is shown relative to eGFP-dsRNA controls. d Viral RNA levels were determined by qRT-PCR using LGTV NS5 primers at 24 h p.i.. The data was normalised to beta actin and is presented for each of the genes listed in the x-axis, and for cells that were not treated with any dsRNA and then infected with LGTV (Virus), as fold changes relative to eGFP dsRNA controls. e Infectious virus present in the supernatant was titrated by plaque assay at 24 h p.i. and the titres are presented for each of the genes listed in the x-axis, and for cells that were not treated with any dsRNA and then infected with LGTV (Virus), as fold change relative to titres in the eGFP-dsRNA control. The mean with standard error of three independent experiments is shown, including only those replicates in which the knockdown was validated. Statistical significance was calculated using two-way ANOVA Fisher’s LSD test (* p < 0.05, ** p < 0.001)