RNA Detection in Urine The Journal of Molecular Diagnostics

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Presentation transcript:

RNA Detection in Urine The Journal of Molecular Diagnostics Mónica Martínez-Fernández, Jesús M. Paramio, Marta Dueñas The Journal of Molecular Diagnostics Volume 18, Issue 1, Pages 15-22 (January 2016) DOI: 10.1016/j.jmoldx.2015.07.008 Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Total RNA mean yield for each of the three conditions from a single urine sample. A: Fresh urine samples. B: After 24 hours frozen at −20°C. C: After storage at room temperature from 0 to 90 days. The mean of each concentration obtained measured by Nanodrop absorbance at 260 nm is inside each bar and percentage coefficient of variation in parentheses. The Journal of Molecular Diagnostics 2016 18, 15-22DOI: (10.1016/j.jmoldx.2015.07.008) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 miRNA, RNU, and mRNA evaluation by specific RT-qPCR at 0 days and after one freeze and thaw cycle at −20°C, extracted using Exiqon (A), Norgen for miRNAs (B), Norgen slurry method (C), Qiagen miRNeasy (D), and a mixed method between slurry and miRNAeasy (E). Light gray bars correspond to fresh samples and dark gray bars correspond to frozen samples. The Journal of Molecular Diagnostics 2016 18, 15-22DOI: (10.1016/j.jmoldx.2015.07.008) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 miRNA, RNU, and mRNA evaluation at different time points, from day 0 to the 3-month period, by specific RT-qPCR of samples stored at room temperature, extracted using the Norgen slurry method (A) and a mixed method between slurry and miRNAeasy (B). Each bar represents the mean of three experimental replicate. The Journal of Molecular Diagnostics 2016 18, 15-22DOI: (10.1016/j.jmoldx.2015.07.008) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Evaluation of daytime variation on RNA purification and qPCR performance. A: Total RNA mean yield for each of the three extractions from a single urine sample at three time points measured by Nanodrop absorbance at 260 nm. B: miRNA, RNU, and mRNA evaluation by specific RT-qPCR from 10 ng of total RNA (B). Gray bars correspond to the Norgen slurry method, and black bars correspond to the mixed method between slurry and miRNAeasy. The Journal of Molecular Diagnostics 2016 18, 15-22DOI: (10.1016/j.jmoldx.2015.07.008) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Evaluation of the five extraction methods on 25 urine samples. Total RNA extracted from each urine sample measured by Nanodrop absorbance at 260 nm (mean of three RNA extractions). Mean yield is given in parentheses, and the upper bars indicate the results of t-test evaluation for each extraction protocol. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. The Journal of Molecular Diagnostics 2016 18, 15-22DOI: (10.1016/j.jmoldx.2015.07.008) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 6 Specific RT-qPCR evaluation of RNA samples purified by the slurry method (A) and mixed protocol (B). Boxplots reveal the distributions of raw Ct values (arithmetic means of triplicates) of the 22 samples for each of the genes tested. Number of samples with Ct values ≤37 are given in parentheses. Ct values >37 were not considered. The Journal of Molecular Diagnostics 2016 18, 15-22DOI: (10.1016/j.jmoldx.2015.07.008) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 7 Representation of both normalizer analyses from Genorm and Normfinder logarithms of the selected genes. Normalizer analysis by GeNorm is based in geometric means of Ct values and Normfinder, a model-based variance estimation approach to identify genes suited for normalization. In both cases, the lower value, the greater stability. The Journal of Molecular Diagnostics 2016 18, 15-22DOI: (10.1016/j.jmoldx.2015.07.008) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions