Bioscience Methods 2025, Vol.16, No.6, 280-288 http://bioscipublisher.com/index.php/bm 284 regulatory relationships cannot be merely guessed. Experimental evidence like qRT-PCR and RACE-PCR has indeed verified the negative regulatory relationship between miRNA and target genes, which can be regarded as solid evidence for these regulatory mechanisms. Figure 2 Co-expression network analysis in well water and water stress treatments. (A) Network visualization in Cytoscape. The nodes were colored by module membership. (B) Correlations between module eigengenes and root phenotypic traits. The numbers within the heatmap represent correlations and p-value (red, positively correlated; green, negatively correlated) for the module–trait associations (SDW, shoot dry weight; SFW, shoot fresh weight; RL, root length; TRL, total root length; Tips, root branches; Forks, root forks; RSA, root surface area; RV, root volume). (C) The connection between zma-miR394b precursor expression and total dry weight. On the left is the root phenotype of some lines from a natural group containing 368 lines. Red means that the expression of zma-miR394b precursor is higher (right) (Adopted from Tang et al., 2022) 5.2 Construction of miRNA-target gene regulatory networks Does one miRNA affect one gene? Sometimes it's really not that simple. Under drought stress, many mirnas and their target genes actually interact in patches and networks. The common practice nowadays is to combine high-throughput sequencing, transcriptome data and co-expression information for analysis, so as to piece together the complete mirNA-target gene network (Sharma et al., 2025). Some "combination punks" are commonly seen in these networks, such as modules like miR156-SPL, miR159-MYB, and miR1119-MYC2, which sometimes carry tissue specificity and genotype differences (Li et al., 2022; Zhakypbek et al., 2025). Methods like WGCNA or degradation groups have also been of great help. They have enabled us to clearly see which mirnas are negatively correlated with the expression of their targets and which ones are in key positions in the drought response.
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