Plasma MicroRNA-21 Is Associated with Tumor Burden in Cutaneous Melanoma  Gerald Saldanha, Linda Potter, Priya Shendge, Joy Osborne, Steve Nicholson, NgiWieh Yii, Sanjay Varma, Muhammad Imran Aslam, Shona Elshaw, Eftychios Papadogeorgakis, J. Howard Pringle  Journal of Investigative Dermatology  Volume 133, Issue 5, Pages 1381-1384 (May 2013) DOI: 10.1038/jid.2012.477 Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 MicroRNA-21 (miR-21) in melanocytic tumor tissue. (a) The relative expression of miR-21 in common acquired nevi (n=13), congenital nevi (n=11), and melanoma (n=51) tissue compared with neonatal normal melanocytes grown in culture. The mean for each group is shown. The results are expressed as ΔΔCT (see Supplementary methods online). (b) MiR-21 C-ISH in a nevus, which is negative. (c) Same nevus but C-ISH for U6, indicating good RNA integrity. (d) MiR-21 C-ISH in a melanoma showing strong staining. Bar in photomicrographs is 1mm. RT-PCR primers are shown in Supplementary Tables S3 and S4 online. Journal of Investigative Dermatology 2013 133, 1381-1384DOI: (10.1038/jid.2012.477) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 MicroRNA-21 (miR-21) in plasma. (a) MiR-21 in paired melanoma and plasma samples. Results are expressed as relative expression; miR-21 expression was normalized to 18s ribosomal RNA to create a ΔCT. Relative expression as 2−ΔΔCT was calculated in relation to the ΔCT of normal human epidermal melanocytes. (b) MiR-21 in paired blood samples taken before and after removal of a melanocytic tumor. Results expressed as ΔCT, relative to miR-191 (see Supplementary methods online). (c) Six categories in which miR-21 was assessed: controls, cancer-free healthy people; N+DN, nevi and dysplastic nevi; MM DF, melanoma patients disease-free for a minimum of 3 years post excision of primary tumor; MM 0+I+II, melanoma patients with primary disease only, i.e., American Joint Committee on Cancer (AJCC) stage 0, I, or II; MM III, melanoma patients with AJCC stage III disease, i.e., regional metastases; and MM IV, melanoma patients with AJCC stage IV disease, i.e., distant metastases. (d) Ranked list of candidate plasma miRNAs after analysis of 740 mature miRNAs and endogenous controls in two matched RNA pools derived from preoperative and postoperative blood samples from patients having regional node resection for stage III melanoma; the list was filtered and ranked by preoperative CT<30 and a fold decrease of >5. (e) miR-211 levels in the same groups as panel c. (f) Cumulative z scores of miR-21 and miR-211, same groups as panel e. Journal of Investigative Dermatology 2013 133, 1381-1384DOI: (10.1038/jid.2012.477) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions