Quantifying BYDV titer and miRNA expression

For quantifying the BYDV titer, cDNA was synthesized using 1 µg of total RNA, random hexamers, and a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer’s instructions. The cDNA was diluted 10-fold for the qPCR assays in a LightCycler 480 (Roche, Basel, Switzerland) using LightCycler 480 SYBR Green I Master (Roche), according to the methods of Jarošová and Kundu [23 (link)]. For confirming the expression of barley miRNAs, cDNA was synthesized using 500 ng of RNA, specific stem-loop primers, and a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). The assay was carried out according to [24 (link)] and multiplexed as described by Turner [25 (link)] (four targeted cDNAs in one reaction). For the normalization strategy adapted from Ferdous [26 (link)] three barley genes were selected (snoR14, snoR23, and hvu-MiR168), and the normalized expression was calculated as an average expression. The primers for cDNA synthesis and for qPCR are given in Supplementary File S1. The relative expression of all miRNAs was calculated using the 2^−ΔΔCt method [27 (link)].