Validation of cold-responsive expression of conserved miRNAs and predicted miRNAs in Brachypodium. Total RNA was isolated from the aerial parts of Brachypodium seedlings with (C) or without (W) cold-treatment and analyzed with RNA gel blots probed with endlabeled antisense oligonucleotides. The 5.8S rRNA bands were visualized by ethidium bromide staining of polyacrylamide gels and served as loading controls. Number in parentheses show the normalized sequencing frequencies of cold-responsive Brachypodium miRNAs in the NC/WC library. Note that the miR911T/926T/927T, miR913T/914T/915T, miR917T/922T/928T and miR918T/919T could not be distinguished because of high sequence similarity.