d16c4fac6fd841329516f8c041391e46 pmc_31562345.pdf 95a6aa902edb15df4ff64855e5f3d209fde90bd8 pmc_31562345.pdf 0a0bfe7b1866a5f6397d42286dad19d4915612ff94053ceea1c8ea9290561dc0 pmc_31562345.pdf Title: Identification of reference genes for RT-qPCR data normalisation in aging studies Subject: Scientific Reports, doi:10.1038/s41598-019-50035-0 Keywords: Author: Lourdes Gonzamp#x000E1;lez-Bermamp#x000FA;dez Creator: Springer Producer: iText® 5.3.5 ©2000-2012 1T3XT BVBA (AGPL-version) CreationDate: Tue Sep 17 13:12:44 2019 CEST ModDate: Mon Sep 23 12:59:16 2019 CEST Tagged: no UserProperties: no Suspects: no Form: AcroForm JavaScript: no Pages: 11 Encrypted: no Page size: 595.276 x 782.362 pts Page rot: 0 File size: 1433084 bytes Optimized: no PDF version: 1.4 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- IYMJBY+Corbel-Bold CID TrueType Identity-H yes yes yes 93 0 VDRPBY+Corbel-BoldItalic CID TrueType Identity-H yes yes yes 501 0 NRYYSO+Corbel-BoldItalic2 Type 1C WinAnsi yes yes yes 502 0 MinionPro-Regular Type 1C Custom yes no yes 95 0 TBSKDW+Corbel-Bold2 Type 1C WinAnsi yes yes yes 96 0 MinionPro-It Type 1C Custom yes no yes 98 0 RXLHQK+Corbel2 Type 1C WinAnsi yes yes yes 99 0 XCDADU+Corbel CID TrueType Identity-H yes yes yes 100 0 MyriadPro-It Type 1C WinAnsi yes no yes 101 0 JFHIKW+TimesNewRomanPSMT Type 1C WinAnsi yes yes yes 503 0 EuclidSymbol Type 1C Custom yes no yes 94 0 EuclidSymbol-Italic Type 1C Custom yes no yes 97 0 MinionPro-Bold Type 1C Custom yes no yes 129 0 GXCRUC+STIXGeneral-Regular Type 1C Custom yes yes yes 153 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2020-08-10 12:25:57 CEST RepresentationInformation: pmc_31562345.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2020-03-02 03:56:15 CET Size: 1433084 Format: PDF Version: 1.4 Status: Well-Formed, but not valid SignatureMatches: PDF-hul ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_50035.indd: 8. Faraonio, R., Pane, F., Intrieri, M., Russo, T. & Cimino, F. In vitro acquired cellular senescence and aging-specific phenotype can be distinguished on the basis of specific mRNA expression. Cell Death Differ. 9, 862–864 (2002). :49' cannot be found Offset: 908326 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_50035.indd:Figure 1. Evaluation of RT-qPCR primer efficiencies. The amplification efficiency for each primer threshold cycle (Ct) and the logarithm of the initial cDNA concentrations were plotted to calculate the slope (S) of each primer pair. Standard cur:78' cannot be found Offset: 908326 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_50035.indd:Figure 2. Ct values of eight candidate reference genes in different aging models and the optimal number of reference genes needed for accurate normalisation calculated by geNorm. Box and whisker plots show the raw Ct values of the candidate ref:84' cannot be found Offset: 908326 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_50035.indd:Figure 2. Ct values of eight candidate reference genes in different aging models and the optimal number of reference genes needed for accurate normalisation calculated by geNorm. Box and whisker plots show the raw Ct values of the candidate re:85' cannot be found Offset: 908326 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_50035.indd:Figure 2. Ct values of eight candidate reference genes in different aging models and the optimal number of reference genes needed for accurate normalisation calculated by geNorm. Box and whisker plots show the raw Ct values of the candidate r:86' cannot be found Offset: 908326 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_50035.indd:Figure 2. Ct values of eight candidate reference genes in different aging models and the optimal number of reference genes needed for accurate normalisation calculated by geNorm. Box and whisker plots show the raw Ct values of the candidate :87' cannot be found Offset: 908326 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_50035.indd:Figure 2. Ct values of eight candidate reference genes in different aging models and the optimal number of reference genes needed for accurate normalisation calculated by geNorm. Box and whisker plots show the raw Ct values of the candidate:88' cannot be found Offset: 908326 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_50035.indd:Figure 2. Ct values of eight candidate reference genes in different aging models and the optimal number of reference genes needed for accurate normalisation calculated by geNorm. Box and whisker plots show the raw Ct values of the candidat:89' cannot be found Offset: 908326 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_50035.indd:Figure 2. Ct values of eight candidate reference genes in different aging models and the optimal number of reference genes needed for accurate normalisation calculated by geNorm. Box and whisker plots show the raw Ct values of the candida:90' cannot be found Offset: 908326 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_50035.indd:Figure 3. Expression levels of target genes normalised to different reference genes. Gene expression levels were normalised to the indicated reference gene or gene pair in the X axis. (a) Relative expression levels of CDKN1A in control and :111' cannot be found Offset: 908326 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_50035.indd:Figure 3. Expression levels of target genes normalised to different reference genes. Gene expression levels were normalised to the indicated reference gene or gene pair in the X axis. (a) Relative expression levels of CDKN1A in control and:112' cannot be found Offset: 908326 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_50035.indd:Figure 3. Expression levels of target genes normalised to different reference genes. Gene expression levels were normalised to the indicated reference gene or gene pair in the X axis. (a) Relative expression levels of CDKN1A in control an:113' cannot be found Offset: 908326 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_50035.indd: 12. de Magalhães, J. P., Curado, J. & Church, G. M. Meta-analysis of age-related gene expression profiles identifies common signatures of aging. Bioinformatics 25, 875–881 (2009). :115' cannot be found Offset: 908326 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_50035.indd:Figure 3. Expression levels of target genes normalised to different reference genes. Gene expression levels were normalised to the indicated reference gene or gene pair in the X axis. (a) Relative expression levels of CDKN1A in control a:117' cannot be found Offset: 908326 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_50035.indd:Figure 3. Expression levels of target genes normalised to different reference genes. Gene expression levels were normalised to the indicated reference gene or gene pair in the X axis. (a) Relative expression levels of CDKN1A in control :118' cannot be found Offset: 908326 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_50035.indd:Figure 3. Expression levels of target genes normalised to different reference genes. Gene expression levels were normalised to the indicated reference gene or gene pair in the X axis. (a) Relative expression levels of CDKN1A in control:119' cannot be found Offset: 908326 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_50035.indd:Figure 3. Expression levels of target genes normalised to different reference genes. Gene expression levels were normalised to the indicated reference gene or gene pair in the X axis. (a) Relative expression levels of CDKN1A in contro:120' cannot be found Offset: 908326 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_50035.indd: 38. Vandesompele, J. et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, RESEARCH0034 (2002). :127' cannot be found Offset: 908326 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_50035.indd: 1. López-Otín, C., Blasco, M. A., Partridge, L., Serrano, M. & Kroemer, G. The Hallmarks of Aging. Cell 153, 1194–1217 (2013). :128' cannot be found Offset: 908326 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_50035.indd: 29. Hernandez-Segura, A., Rubingh, R. & Demaria, M. Identification of stable senescence-associated reference genes. Aging Cell 18, e12911 (2019). :133' cannot be found Offset: 908326 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_50035.indd: 27. Touchberry, C. D., Wacker, M. J., Richmond, S. R., Whitman, S. A. & Godard, M. P. Age-related changes in relative expression of real-time PCR housekeeping genes in human skeletal muscle. J. Biomol. Tech. 17, 157–62 (2006). :147' cannot be found Offset: 908326 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_50035.indd: 30. de Jonge, H. J. M. et al. Evidence Based Selection of Housekeeping Genes. PLoS One 2, e898 (2007). :152' cannot be found Offset: 908326 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_50035.indd: 42. Bustin, S. A. et al. The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Clin. Chem. 55, 611–22 (2009). :154' cannot be found Offset: 908326 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'SpringerNature_SciRep_50035.indd: 37. Andersen, C. L., Jensen, J. L. & Ørntoft, T. F. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon ca:156' cannot be found Offset: 908326 MIMEtype: application/pdf PDFMetadata: Objects: 550 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: false CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseThumbs Language: EN Outlines: Item: Title: Identification of reference genes for RT-qPCR data normalisation in aging studies Children: Item: Title: Results Children: Item: Title: Establishment of senescence and aging models and testing of primers for reference genes. Item: Title: Descriptive statistics of candidate reference genes. Item: Title: Expression stability of candidate reference genes. Item: Title: Validation of selected reference genes. Item: Title: Discussion Item: Title: Materials and Methods Children: Item: Title: Cell culture. Item: Title: Senescence induction and detection. Item: Title: RNA extraction and cDNA synthesis. Item: Title: Selection of reference genes and primers design. Item: Title: Quantitative PCR and primer efficiency calculations. Item: Title: Data analysis. Item: Title: Validation of candidate reference genes. Item: Title: Statistical analysis. Item: Title: Ethical statement. Item: Title: Acknowledgements Item: Title: Figure 1 Evaluation of RT-qPCR primer efficiencies. Item: Title: Figure 2 Ct values of eight candidate reference genes in different aging models and the optimal number of reference genes needed for accurate normalisation calculated by geNorm. Item: Title: Figure 3 Expression levels of target genes normalised to different reference genes. Item: Title: Table 1 List of the reference genes tested by RT-qPCR. Item: Title: Table 2 Candidate reference gene expression stability ranked by NormFinder and geNorm in different aging processes. 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XMP: application/pdf doi:10.1038/s41598-019-50035-0 Springer US Scientific Reports, doi:10.1038/s41598-019-50035-0 Identification of reference genes for RT-qPCR data normalisation in aging studies Lourdes Gonzamp#x000E1;lez-Bermamp#x000FA;dez Teresa Anglada Anna Genescamp#x000E0; Marta Martamp#x000ED;n Mariona Terradas 10.1038/s41598-019-50035-0 http://dx.doi.org/10.1038/s41598-019-50035-0 journal Scientific Reports © 2019, The Author(s) 2045-2322 10.1038/s41598-019-50035-0 Springer 2019-09-23T12:59:16+02:00 2019-09-17T16:42:44+05:30 2019-09-23T12:59:16+02:00 True iText® 5.3.5 ©2000-2012 1T3XT BVBA (AGPL-version) VoR uuid:a0f442af-34b5-4718-a4e0-7f314832c30e uuid:cca5d4fc-f0f9-4054-b11d-bf8b08d4f5ae default 1 converted uuid:a7a50453-1662-460f-9dc1-463dbf4893e0 converted to PDF/A-2b pdfToolbox 2019-09-17T16:43:30+05:30 2 B http://ns.adobe.com/pdf/1.3/ pdf Adobe PDF Schema internal A name object indicating whether the document has been modified to include trapping information Trapped 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