Validation of Circulating miRNA Biomarkers for Predicting Lymph Node Metastasis in Gastric Cancer  Shine Young Kim, Tae Yong Jeon, Chang In Choi, Dae Hwan Kim, Dong Heon Kim, Gwang Ha Kim, Dong Yup Ryu, Bong Eun Lee, Hyung Hoi Kim  The Journal of Molecular Diagnostics  Volume 15, Issue 5, Pages 661-669 (September 2013) DOI: 10.1016/j.jmoldx.2013.04.004 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Standard curves of miR-21, miR-27a, miR-106b, miR-223, miR-146a, miR-148a, and miR-433 using synthetic miRNAs. Tenfold serial dilutions of synthetic miRNAs were used. The initial dilution rates were adjusted so that a maximally diluted sample could be detected within 40 CT cycles. Linearity was confirmed within concentrations from 200 to 0.002 amol/μL: miR-21, y = −3.4969x − 1.8946 (R2 = 0.9995); miR-27a, y = −3.242x + 2.7904 (R2 = 0.9991); miR-106b, y = −3.3428x − 0.8143 (R2 = 0.9995); miR-223, y = −3.5909x − 2.2419 (R2 = 0.9998); miR-146a, y = −3.2604x + 2.6641 (R2 = 0.9988); miR-148a, y = −3.3035x + 2.3569 (R2 = 0.9998); and miR-433, y = −3.7469x − 3.1172 (R2 = 0.9981). The Journal of Molecular Diagnostics 2013 15, 661-669DOI: (10.1016/j.jmoldx.2013.04.004) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Box plots of serum miRNA concentrations in patients with GC and controls. The expression levels of six miRNAs (miR-21, miR-27a, miR-106b, miR-146a, miR-148a, and miR-223) were up-regulated significantly in the LNpGC group compared with those in the LNnGC group. We confirmed that miR-106b and miR-223 expression levels in the 31 patients with GC were up-regulated compared with those in the healthy group. The Journal of Molecular Diagnostics 2013 15, 661-669DOI: (10.1016/j.jmoldx.2013.04.004) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 The ROC curve analysis in the miRNA assay for detecting LN metastasis in patients with GC in a pilot study. The Journal of Molecular Diagnostics 2013 15, 661-669DOI: (10.1016/j.jmoldx.2013.04.004) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Concentrations of serum miRNAs by nodal stage. A: There was a tendency that higher serum miRNA levels were observed with higher nodal stage (N = 79). B: pN1 showed much higher miRNA levels in pT1 patients than those in pN0 patients (N = 19). Asterisks denote extreme outliers; open circles, mild outliers. The Journal of Molecular Diagnostics 2013 15, 661-669DOI: (10.1016/j.jmoldx.2013.04.004) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 ROC curve analysis in the miRNA assay for detecting LN metastasis in patients with GC in the validation study. The combinations of each miRNA were performed by simple summation of each miRNA concentration. The Journal of Molecular Diagnostics 2013 15, 661-669DOI: (10.1016/j.jmoldx.2013.04.004) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions