A Strategy to Find Suitable Reference Genes for miRNA Quantitative PCR Analysis and Its Application to Cervical Specimens Iris Babion, Barbara C. Snoek,

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A Strategy to Find Suitable Reference Genes for miRNA Quantitative PCR Analysis and Its Application to Cervical Specimens Iris Babion, Barbara C. Snoek, Mark A. van de Wiel, Saskia M. Wilting, Renske D.M. Steenbergen The Journal of Molecular Diagnostics Volume 19, Issue 5, Pages 625-637 (September 2017) DOI: 10.1016/j.jmoldx.2017.04.010 Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Strategy for the selection of reference genes for miRNA qPCR normalization. The Journal of Molecular Diagnostics 2017 19, 625-637DOI: (10.1016/j.jmoldx.2017.04.010) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Expression levels of candidate reference genes and spike-in miRNA in cervical specimens. The expression levels are represented by PCR cycle threshold numbers (Cq values). High Cq values indicate low expression and vice versa. Three types of cervical specimens were included: cervical tissues (A), cervical scrapes (B), self-samples (C), and all samples combined (D). Box plots show medians with lower and upper quartiles, and range whiskers. Dashed lines indicate the upper quartile (75% of data) cutoff at 31 Cq. Candidate reference genes that exhibited an upper quartile value higher than 31 Cq were excluded from further analysis (dark gray). The Journal of Molecular Diagnostics 2017 19, 625-637DOI: (10.1016/j.jmoldx.2017.04.010) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Correlation between the two biologically most stable reference genes in cervical tissues (A), cervical scrapes (B), and self-samples (C). The expression levels are represented by Cq values. Pearson correlation coefficient (r) values are shown. CIN, cervical intraepithelial neoplasia; SCC, squamous cell carcinoma. The Journal of Molecular Diagnostics 2017 19, 625-637DOI: (10.1016/j.jmoldx.2017.04.010) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Validation of selected reference genes in cervical specimens. Three types of cervical specimens were tested, including cervical tissues (A), cervical scrapes (B), and self-samples (C). The relative quantification of miR-15b and miR-100 was assessed using a combination of two commonly used reference genes (U6 + RNU6B) and two biologically most stable reference genes (tissues: RNU24 + miR-423; scrapes: RNU43 + miR-423; self-samples: miR-423 + miR-30b). Normal samples are marked in green, cervical intraepithelial neoplasia (CIN) 2/3 in orange, and squamous cell carcinomas (SCCs) in red. Box plots show medians with lower and upper quartiles, and range whiskers. All values were log2 transformed and subsequently centered to obtain values on the same scale. The Journal of Molecular Diagnostics 2017 19, 625-637DOI: (10.1016/j.jmoldx.2017.04.010) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Analysis of P values and signal-to-noise ratios (SNRs) between normal, cervical intraepithelial neoplasia (CIN) 2/3, and squamous cell carcinoma (SCC) cervical specimens. No normalization, normalization using two commonly used reference genes (U6 +RNU6B), and normalization using the two biologically most stable reference genes (tissues: RNU24 + miR-423; scrapes: RNU43 + miR-423; self-samples: miR-423 + miR-30b) were evaluated. P values were computed by a two-sample t-statistic on log2 transformed expression data for miR-15b (A) and miR-100 (B). The black horizontal lines indicate significance level 0.05. SNRs for miR15b (C) and miR-100 (D) are shown. Positive SNRs indicate up-regulation, and negative ratios correspond to down-regulation. The Journal of Molecular Diagnostics 2017 19, 625-637DOI: (10.1016/j.jmoldx.2017.04.010) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S1 Candidate reference genes selected from small RNA sequencing data in self-samples (A) and miRNA microarray data obtained from cervical tissues (B). Previously described reference genes that were excluded based on small RNA sequencing data in self-samples (C) and miRNA microarray data in tissues (D). CIN, cervical intraepithelial neoplasia; SCC, squamous cell carcinoma. The Journal of Molecular Diagnostics 2017 19, 625-637DOI: (10.1016/j.jmoldx.2017.04.010) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S2 Optimal number of reference genes for normalization determined by NormFinder in cervical tissues (A), cervical scrapes (B), and self-samples (C). The Journal of Molecular Diagnostics 2017 19, 625-637DOI: (10.1016/j.jmoldx.2017.04.010) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions