3.6 qRT-PCR analysis
To further analyze the hypoxia-induced biological processes in P. vachelli muscles, we performed qRT-PCR analysis on selected 16 DEMI and 70 DEG. These representative miRNAs/genes were artificially selected to determine their potential role in environmental hypoxia for P. vachelli muscles. For example, all 16 DEMI (miR-15b/c, 133b, 181a, 193b, 338, 457a, 1-2, 18a, 30a/d, 210, 214, PC-7236, let-7b) were from the multi-omics identification (Fig. 5 ) and some DEGs were related to important biological processes (i.e., oxygen sensor, angiogenesis, heme and erythropoiesis, glycolysis, TCA cycle, lipolysis, oxidative stress, mitochondrial dysfunction, NOD-like receptor, NF-kappa B, MAPK signaling pathway, cell cycle, and apoptosis). The results of qRT-PCR revealed similar expression patterns between most of these mRNAs/miRNAs and those from transcriptome/miRNAome data (TPM/reads-based expression values) (Table 1; Fig. 5; Fig. 6 ). Of these, miR-15c showed minimal expression in control groups (M0a, M0b, M0c) in the qRT-PCR analysis, and was induced by hypoxia stress, which was consistent with the present miRNA-seq data. Although there were some quantitative differences between the two analysis platforms, our results showed similar trends between the qRT-PCR and RNA-seq data.