Presentation is loading. Please wait.

Presentation is loading. Please wait.

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

Published byKenneth Jordan Modified over 6 years ago

Similar presentations

Presentation on theme: "A Strategy to Find Suitable Reference Genes for miRNA Quantitative PCR Analysis and Its Application to Cervical Specimens  Iris Babion, Barbara C. Snoek,"— Presentation transcript:

1 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 (September 2017) DOI: /j.jmoldx Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

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

3 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  , DOI: ( /j.jmoldx ) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4 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  , DOI: ( /j.jmoldx ) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5 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 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  , DOI: ( /j.jmoldx ) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

6 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 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 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  , DOI: ( /j.jmoldx ) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

7 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  , DOI: ( /j.jmoldx ) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

8 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  , DOI: ( /j.jmoldx ) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

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

Similar presentations

Telomerase Activity and Expression of Telomerase RNA Component and Telomerase Catalytic Subunit Gene in Cervical Cancer Kenji Nakano, Elizabeth Watney,

Telomerase Activity and Expression of Telomerase RNA Component and Telomerase Catalytic Subunit Gene in Cervical Cancer Kenji Nakano, Elizabeth Watney,
Design and Multiseries Validation of a Web-Based Gene Expression Assay for Predicting Breast Cancer Recurrence and Patient Survival Ryan K. Van Laar The.

Design and Multiseries Validation of a Web-Based Gene Expression Assay for Predicting Breast Cancer Recurrence and Patient Survival Ryan K. Van Laar The.
Hepatitis C Virus RNA Real-Time Quantitative RT-PCR Method Based on a New Primer Design Strategy  Lida Chen, Wenli Li, Kuo Zhang, Rui Zhang, Tian Lu,

Hepatitis C Virus RNA Real-Time Quantitative RT-PCR Method Based on a New Primer Design Strategy  Lida Chen, Wenli Li, Kuo Zhang, Rui Zhang, Tian Lu,
Molecular Analysis of the Breast Cancer Genes BRCA1 and BRCA2 Using Amplicon- Based Massive Parallel Pyrosequencing  Geneviève Michils, Silke Hollants,

Molecular Analysis of the Breast Cancer Genes BRCA1 and BRCA2 Using Amplicon- Based Massive Parallel Pyrosequencing  Geneviève Michils, Silke Hollants,
Comparison of Clinical Targeted Next-Generation Sequence Data from Formalin-Fixed and Fresh-Frozen Tissue Specimens  David H. Spencer, Jennifer K. Sehn,

Comparison of Clinical Targeted Next-Generation Sequence Data from Formalin-Fixed and Fresh-Frozen Tissue Specimens  David H. Spencer, Jennifer K. Sehn,
Anna Sapino, Paul Roepman, Sabine C. Linn, Mireille H. J

Anna Sapino, Paul Roepman, Sabine C. Linn, Mireille H. J
Hepatitis C Virus RNA Real-Time Quantitative RT-PCR Method Based on a New Primer Design Strategy  Lida Chen, Wenli Li, Kuo Zhang, Rui Zhang, Tian Lu,

Hepatitis C Virus RNA Real-Time Quantitative RT-PCR Method Based on a New Primer Design Strategy  Lida Chen, Wenli Li, Kuo Zhang, Rui Zhang, Tian Lu,
Droplet Digital PCR for Absolute Quantification of EML4-ALK Gene Rearrangement in Lung Adenocarcinoma  Qiushi Wang, Xin Yang, Yong He, Qiang Ma, Li Lin,

Droplet Digital PCR for Absolute Quantification of EML4-ALK Gene Rearrangement in Lung Adenocarcinoma  Qiushi Wang, Xin Yang, Yong He, Qiang Ma, Li Lin,
Accurate Molecular Classification of Renal Tumors Using MicroRNA Expression  Eddie Fridman, Zohar Dotan, Iris Barshack, Miriam Ben David, Avital Dov, Sarit.

Accurate Molecular Classification of Renal Tumors Using MicroRNA Expression  Eddie Fridman, Zohar Dotan, Iris Barshack, Miriam Ben David, Avital Dov, Sarit.
Bead Array–Based microRNA Expression Profiling of Peripheral Blood and the Impact of Different RNA Isolation Approaches  Andrea Gaarz, Svenja Debey-Pascher,

Bead Array–Based microRNA Expression Profiling of Peripheral Blood and the Impact of Different RNA Isolation Approaches  Andrea Gaarz, Svenja Debey-Pascher,
Laila C. Schenkel, Charles Schwartz, Cindy Skinner, David I

Laila C. Schenkel, Charles Schwartz, Cindy Skinner, David I
Morten Andersen, Morten Grauslund, Jesper Ravn, Jens B

Morten Andersen, Morten Grauslund, Jesper Ravn, Jens B
Optimization of a Relative Telomere Length Assay by Monochromatic Multiplex Real- Time Quantitative PCR on the LightCycler 480  Anthony Y.Y. Hsieh, Sara.

Optimization of a Relative Telomere Length Assay by Monochromatic Multiplex Real- Time Quantitative PCR on the LightCycler 480  Anthony Y.Y. Hsieh, Sara.
MicroRNAs in spent blastocyst culture medium are derived from trophectoderm cells and can be explored for human embryo reproductive competence assessment 

MicroRNAs in spent blastocyst culture medium are derived from trophectoderm cells and can be explored for human embryo reproductive competence assessment 
A Quantitative Allele-Specific PCR Test for the BRAF V600E Mutation Using a Single Heterozygous Control Plasmid for Quantitation  Philippe Szankasi, N.

A Quantitative Allele-Specific PCR Test for the BRAF V600E Mutation Using a Single Heterozygous Control Plasmid for Quantitation  Philippe Szankasi, N.
Separate Quality-Control Measures Are Necessary for Estimation of RNA and Methylated DNA from Formalin-Fixed, Paraffin-Embedded Specimens by Quantitative.

Separate Quality-Control Measures Are Necessary for Estimation of RNA and Methylated DNA from Formalin-Fixed, Paraffin-Embedded Specimens by Quantitative.
A Single-Tube Multiplexed Assay for Detecting ALK, ROS1, and RET Fusions in Lung Cancer  Maruja E. Lira, Yoon-La Choi, Sun Min Lim, Shibing Deng, Donghui.

A Single-Tube Multiplexed Assay for Detecting ALK, ROS1, and RET Fusions in Lung Cancer  Maruja E. Lira, Yoon-La Choi, Sun Min Lim, Shibing Deng, Donghui.
Christos Sotiriou, Chand Khanna, Amir A

Christos Sotiriou, Chand Khanna, Amir A
Simple Detection of Telomere Fusions in Pancreatic Cancer, Intraductal Papillary Mucinous Neoplasm, and Pancreatic Cyst Fluid  Tatsuo Hata, Marco Dal.

Simple Detection of Telomere Fusions in Pancreatic Cancer, Intraductal Papillary Mucinous Neoplasm, and Pancreatic Cyst Fluid  Tatsuo Hata, Marco Dal.
A Multiplex SNaPshot Assay as a Rapid Method for Detecting KRAS and BRAF Mutations in Advanced Colorectal Cancers  Sandrine Magnin, Erika Viel, Alice.

A Multiplex SNaPshot Assay as a Rapid Method for Detecting KRAS and BRAF Mutations in Advanced Colorectal Cancers  Sandrine Magnin, Erika Viel, Alice.

Similar presentations

Ads by Google