Quantification of Circulating miRNAs in Plasma Ioanna S. Sourvinou, Athina Markou, Evi S. Lianidou  The Journal of Molecular Diagnostics  Volume 15, Issue 6, Pages 827-834 (November 2013) DOI: 10.1016/j.jmoldx.2013.07.005 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Isolation of plasma circulating miRNAs using TRIzol LS reagent. Raw Cq values for endogenous hsa-miR-21 after total RNA extraction, exogenous cel-miR-39 added in sample before total RNA extraction, and exogenous cel-miR-39 recovered after extraction are shown starting from different plasma volumes (A) and as the effect of temperature and time of the precipitation step (B). The data represent the means ± SD of three independent experiments. Statistical evaluation was based on the U-test. ∗P < 0.05. The Journal of Molecular Diagnostics 2013 15, 827-834DOI: (10.1016/j.jmoldx.2013.07.005) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 A: RT-qPCR raw Cq values for endogenous hsa-miR-21 after total RNA extraction, exogenous cel-miR-39 added in sample before total RNA extraction, and exogenous cel-miR-39 recovered after extraction from plasma samples using different commercially available extraction protocols. B: Recovery rates for the exogenous spiked-in control cel-miR-39. The data represent the means ± SD of three independent experiments. Statistical evaluation was based on the U-test. ∗P < 0.05. The Journal of Molecular Diagnostics 2013 15, 827-834DOI: (10.1016/j.jmoldx.2013.07.005) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Stability study of circulating miRNAs in plasma at different storage time and temperature conditions. A and B: cel-miR-39. C and D: hsa-miR-21. E and F: hsa-miR-16. The results are presented as differences in raw Cq values from the 0-hour control (0h) (positive differences represent decreases and negative differences represent increases in miRNA concentrations) and as percentage recoveries from the 0h for each miRNA. The data represent the means ± SD of three independent experiments. Statistical evaluation was based on the U-test. ∗P < 0.05. The Journal of Molecular Diagnostics 2013 15, 827-834DOI: (10.1016/j.jmoldx.2013.07.005) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Percentage recovery for exogenous cel-miR-39 before and after storage of extracted miRNAs for 2, 6, 8, 9, and 12 months. The data represent the means of three independent measurements. The Journal of Molecular Diagnostics 2013 15, 827-834DOI: (10.1016/j.jmoldx.2013.07.005) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Effects of different normalization procedures on the quantification of circulating miRNA levels in the plasma of healthy individuals (n = 30) and patients with NSCLC (n = 30). The concentration of hsa-miR-21 in plasma was quantified by normalizing with respect to hsa-miR-16 (A), exogenous control cel-miR-39 (B), and a combination of both (C). ∗∗∗P < 0.001. The Journal of Molecular Diagnostics 2013 15, 827-834DOI: (10.1016/j.jmoldx.2013.07.005) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions