Fig. 5

RT-qPCR validation of the top 15 differentially expressed miRNAs between Oncotype DX® DCIS risk groups based on expression levels and age of patients. a Five Oncotype DX® DCIS risk score-related miRNAs, six age-related miRNAs, and four miRNAs based on a combination of Oncotype DX® DCIS risk score-related and patient age-related, identified by NGS analyses, were selected for RT-qPCR validations. b A positive correlation was observed for log2-transformed small-RNA sequencing read counts and RT-qPCR Ct values obtained for the top 15 differentially expressed miRNAs evaluated using RNA samples from DCIS lesions classified with Oncotype DX DCIS assay (see Fig. 2, RT-qPCR samples (n = 30). c Individual box plot evaluations of the 15 selected miRNAs measured by RT-qPCR in 30 samples (See Fig. 2), between low and high/intermediate Oncotype DX® DCIS risk score groups for identification of the top 5 differentially expressed miRNAs between the two groups (miR-135a, miR-190b, miR-205, miR-30c and miR-744). d Composite RT-qPCR miRNA score based on RT-qPCR expression of the 5 differentially expressed miRNAs. A highly significant expression difference (p < 0.0017) was obtained between the two Oncotype DX® DCIS risk groups (low and intermediate/high groups). The red dashed rectangle, in the low composite miRNA group (left), identifies DCIS specimen (i.e., Oncotype DX® DCIS low-risk score of 33 in Table 1) from patient DCIS-L7 who experienced an ipsilateral DCIS recurrence after 2 years of the initial DCIS diagnosis. The blue dashed rectangle, in the Int-High miRNA group (right), identifies DCIS specimen (i.e., Oncotype DX® DCIS intermediate risk score of 40 in Table 1), from the patient DCIS-I9 who experienced an ipsilateral invasive breast cancer recurrence after 2 years of the initial DCIS diagnosis