Separate Quality-Control Measures Are Necessary for Estimation of RNA and Methylated DNA from Formalin-Fixed, Paraffin-Embedded Specimens by Quantitative.

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Separate Quality-Control Measures Are Necessary for Estimation of RNA and Methylated DNA from Formalin-Fixed, Paraffin-Embedded Specimens by Quantitative PCR Aruna Korlimarla, Jyothi S. Prabhu, C.E. Anupama, Jose Remacle, Kanu Wahi, T.S. Sridhar The Journal of Molecular Diagnostics Volume 16, Issue 2, Pages 253-260 (March 2014) DOI: 10.1016/j.jmoldx.2013.11.003 Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Transcript abundance was plotted against ER protein levels estimated by Allred score. The ROC graph was generated using an Allred score of ≥2 as ER+ and a score of 0 as ER−. These data were plotted against ESR1 relative normalized units (RNU). The 95% confidence intervals are indicated by gray curves bracketing the ROC curve. A: All specimens (n = 250). AUC = 0.825. Best cutoff = 9.62. Sensitivity = 76.69% (95% CI, 69.4% to 82.9%). Specificity = 79.31% (95% CI, 69.3% to 87.3%). B: Specimens with well-preserved RNA. AUC = 0.91. Best cutoff = 8.24. Sensitivity = 91.67% (95% CI, 85.9% to 95.6%). Specificity = 79.31% (95% CI, 69.3% to 87.3%). C: Specimens with degraded RNA. AUC = 0.67. Best cutoff = 10.23. Sensitivity = 84.21% (95% CI, 60.4% to 96.6%). Specificity = 54.55% (95% CI, 32.2% to 75.6%). The Journal of Molecular Diagnostics 2014 16, 253-260DOI: (10.1016/j.jmoldx.2013.11.003) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Quality of DNA versus quality of RNA. Distribution of CT values for ALU-C4 DNA sequences in a TaqMan assay, comparing specimens with well-preserved versus poorly preserved RNA. Specimens with adequate RNA (n = 122) had a lower median CT value of 19.9, compared with specimens with poorly preserved RNA (n = 27), which had a median value of 21.6. The Journal of Molecular Diagnostics 2014 16, 253-260DOI: (10.1016/j.jmoldx.2013.11.003) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 The relationship between DNA adequacy for MethyLight assay and RNA adequacy for quantitative estimations of transcript abundance in the same FFPE specimen. ALU-C4 CT values are plotted against the RG mean CT values for the same specimen. The quadrants are established by the cutoff values (intersecting lines) for the respective assays. The percentage of specimens in each quadrant is presented below the x axis. n =149. The Journal of Molecular Diagnostics 2014 16, 253-260DOI: (10.1016/j.jmoldx.2013.11.003) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions