Northern Blotting of Viral siRNAs

Northern blotting was used to validate the presence of vsiRNAs. Briefly, 2 μg of sRNA from virus-free and virus-infected A. fumigatus isolates were fractionated by electrophoresis through 7 M urea 16% denaturing polyacrylamide gels. Following electrophoresis the RNAs were transferred onto Hybond NX membrane (Amersham) using the semi-dry transfer system (Bio-Rad). Cross-linking was performed using 1-ethyl-3-(dimethylaminopropyl) carbodiimide at 60 °C for 2 h as described previously [45 (link)]. Hybridisation was done at 37 °C overnight using P³² labelled probes which were reverse complement oligonucleotides to the sRNAs of interest. To assess the relative expression levels, quantifications were done three times using ImageQuant software (version 8.1, GE Healthcare Life Sciences) and averages were plotted. The sequences of the probes used and validated sRNAs are listed in Additional file 4.