Two separate approaches (discrete temporal association, hierarchical clustering) were used to uncover the temporal relationship(s) between microRNAs and their downstream targets in each brain region. Both methods used the "microRNA target filter" utility in Ingenuity Pathway Analysis (IPA, September 2015 release, Qiagen Redwood City, www.qiagen.com/ingenuity) to associate microRNAs with their previously reported DE mRNA targets [26]. It should be noted that microRNAs and mRNAs were isolated from the same samples. Data from mRNA analyses were deposited in the NCBI Gene Expression Omnibus (GEO) under accession number GSE 60676. Only experimentally observed microRNA-target interactions and those predicted with high confidence were used. IPA assigns "high confidence" to interactions involving a conserved or highly conserved microRNA as defined by TargetScan [34–37] and at least one conserved site on the targeted sequence or having a TargetScan total context score of -0.4 or less.
