Research Paper Volume 11, Issue 10 pp 2968—2997

Figure 4. The expression of putative mRNA target genes of endo-siRNAs differentially abundant in young and aged oocytes. To verify that the changes in endo-siRNA abundance led to altered target gene expression in aged oocytes, six mRNAs were selected for RT-qPCR assessment. Candidate mRNAs included three representatives with targeting endo-siRNAs whose abundance was upregulated in aged oocytes (Kifc1, Kifc5b, and Zcchc3), two that were potentially targeted by downregulated endo-siRNAs (Gpr149 and Sp110) and one mRNA potentially targeted by an endo-siRNA with unchanged abundance in mRNA potentially targeted by an endo-siRNA with unchanged abundance in between young and aged oocytes (Oog4). cDNA generation and RT-qPCR experiments were performed in technical and biological triplicate, with each biological replicate comprising 10 oocytes randomly sampled from a pool of oocytes isolated from three animals. Ppia was employed as an endogenous control to normalize the expression levels of target mRNAs. Values are shown as a mean of all replicates ± SEM. Statistical analyses were performed using Student’s t-test, * p < 0.05, *** p < 0.001. Log₂ fold changes based on RNA-Seq are represented as pink, red, and orange line graphs while the relative abundance (2^-ΔCt) of each sRNA determined by RT-qPCR is represented by columns.