Characterization of Hypoxia Induced Maize miRNAs Figure 5. miRNA expression profile by SLRT-PCR and cluster analysis. A: Model of miRNA expression pattern. B: Heatmap of miRNA expression profile. The cluster was done on the basis of log2 (expression level in treatment/expression level in control). Yellow shows down- regulation. Blue shows up-regulation. doi:10.1371/journal.pone.0039786.g005 5-prime RNA Ligase Mediated Rapid Amplification of cDNA Ends (59-RLM-RACE) Validation of Selected miRNA Targets 59-RLM-RACE was preformed on thirteen selected miRNA targets to identify their miRNA cleavage site. These 13 targets are targeted by 8 miRNA families (miR159, miR164, miR167, miR172, miR319, miR393, miR528, miRn7) (Table S9), which are active participants in the signal transduction at the early stage of hypoxia conditions. There are six targets (two GAMYB targets of miR159/319, two ARF targets of miR167, one AP2/ERF target of miR172, and one F-box (TIR1-like gene)), which were validated by the 59-RLM-RACE (Figure 8). Furthermore, the negative correlation expressions between miRNAs and their targets also imply their relationship, which suggested that those miRNA targets were regulated by their miRNAs. Cis-regulatory Element Analysis of miRNA Promoters in B73 To ensure reliable predictions of cis-regulatory elements, also called as transcription factor binding sites (TFBS) or motifs, differentially expressed miRNAs with known transcription start sites (TSS) were used for promoter analysis. To get TSS, we mapped the pre-miRNA coordinates of differentially expressed miRNAs from miRBase release 18 [32] to their B73 RefGen v1 chromosomal locations [27]. Out of 64 differentially expressed miRNA genes representing 22 known mature miRNA signatures; we could map the TSS only for 30 miRNA genes belonging to 13 miRNA families (Table S10). We further confirmed the coordi- nates of 16 pri-miRNA genes with published 59 RACE data [11]. The promoter region [-1000,+500 with respect to TSS] of these 30 differentially expressed miRNA genes were used for computational motif predictions using all experimentally derived position weight matrices (PWMs) from JASPAR core Plantae (http://jasper. PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e39786