Abstract <p>Investigating fungal gene expression in complex solid-state fermentation (SSF) systems is essential for process optimization but is hindered by challenges in RNA extraction. RNA extraction from fungal samples rich in plant biomass is a special challenge that is not met by the commercial RNA extraction kits and conventional RNA extraction methods we have tested. This study presents an optimized RNA extraction method, suitable for RT-qPCR from agricultural side streams fermented by filamentous fungi. The method was applied to investigate the temporal expression of the class II hydrophobin NC2 in <i>Neurospora intermedia</i> grown on four different sources of brewer’s spent grain (BSG): IPA, Pilsner, Stout, and a microbrewery batch. Relative NC2 expression showed a clear temporal increase, peaking on day 6, with the microbrewery BSG batch having the highest peak expression. It demonstrates the applicability of the presented RNA extraction method to monitor expression of a gene of interest during solid-state fermentation of a complex substrate. Furthermore, the RNA extraction protocol was successfully validated on other challenging substrates, including rapeseed press cake and oat hulls, and with other fungi (<i>Aspergillus oryzae</i> and <i>Trichoderma asperellum</i>), consistently yielding high-purity RNA. This work provides a reliable method for RNA extraction from complex SSF products that can be used for gene expression analysis.</p> Key points <p>• <i>Reliable RNA extraction protocol developed for complex fungal SSF substrates</i></p> <p>• <i>Hydrophobin NC2 expression in N. intermedia might be temporally regulated on BSG</i></p> <p>• <i>Peak NC2 expression (day 6) was observed on BSG from a microbrewery</i></p>