Forward (F) and reverse (R) primers for MAP3K3 (XM_015299375, F: 5'-TGA CCC GGA AAT ACA CTC GC; R: 5'-ACA TTC CCA GCA GAG TCA CG), MAP3K2 (XM_015290042, F: 5'-TGA GGG ACC CAC CAG AAA GA; R: 5'-GCG AGT CAC GTT CTC TGT CA), AQP4 (NM_001004765, F: 5'-GTG CCA GTA TGA ACC CTG CT; R: 5'-AAG AGC ACC AGC AAG GAC TG), and IGF2BP3 (NM_001006359, F: 5'-GCC TTG GCA GTT GGA GCT AT; R: 5'-AGC TTG GCA TCT GGT CCT TC) were designed using the Primer3 program (National Center for Biotechnology Information [NCBI] database) and synthesized by Genotech Co.
The miRNA gga-miR-9-5p targets 260 genes, with the top-ranking/ top-scoring gene being MAP3K3 (target rank 1, score 100).
Real-time qPCR was used to further examine the expression of the four miRNAs (gga-miR-9-5p, gga-miR-20b-5p, gga-miR196-5p, and gga-let-7d) that showed the highest fold-changes in the IML of NE-induced chickens, compared to their respective controls, as well as the expression of their top-ranking target genes (MAP3K3, MAP3K2, AQP4, and IGF2BP3, respectively) (Figure 2).
This hypothesis is somewhat supported in the case of gga-miR-9-5p, gga-miR-20b-5p, gga-miR-196-5p, and gga-let-7d and the expression of their target genes MAP3K3, MAP3K2, AQP4, and IGF2BP3, respectively in the IML of NE-induced M5.1 chickens.
