Validation and clustering analysis of developmentally regulated miRNAs. A-C. The expression of three groups of miRNAs at different developmental stages revealed by deep-sequencing. The three groups are: gradual increasing (A), gradual decreasing (B), and peak at middle stages (C). The expression level of miRNAs (TPM) at each developmental stage was normalized to that at E10. D-F. Quantitative PCR (qPCR) analysis of the expression profile of different groups of miRNAs shown in A-C. Results are based on average of three independent experiments (Mean ± SD). The developmental changes of the expression of most miRNAs revealed by qPCR are consistent with results from deep-sequencing. G. Clustering of differentially expressed microRNAs. The Complete Linkage Clustering was used by R package based on expression levels (TPM) of each miRNA at different stages. Both known miRNAs and novel miRNAs are included (DatasetS1). Red means highly expressed and green means lowly expressed.