Valid gene expression normalization by RT-qPCR in studies on hPDL fibroblasts with focus on orthodontic tooth movement and periodontitis

URN to cite this document:

urn:nbn:de:bvb:355-epub-362120 Copy

DOI to cite this document:

10.5283/epub.36212 Copy

Kirschneck, Christian ; Batschkus, Sarah ; Proff, Peter ; Köstler, Josef ; Spanier, Gerrit ; Schröder, Agnes

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Date of publication of this fulltext: 22 Jan 2018 11:26

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Details

Item type:	Article
Journal or Publication Title:	Scientific Reports
Publisher:	NATURE PUBLISHING GROUP
Place of Publication:	LONDON
Volume:	2017
Number of Issue or Book Chapter:	7
Page Range:	pp. 1-13
Date:	7 November 2017
Institutions:	Medicine > Lehrstuhl für Kieferorthopädie Medicine > Lehrstuhl für Medizinische Mikrobiologie und Hygiene Medicine > Lehrstuhl für Mund-, Kiefer- und Gesichtschirurgie
Projects:	Open Access Publizieren (DFG)
Identification Number:	Value Type 10.1038/s41598-017-15281-0 DOI Article number: 14751 Other
Keywords:	REAL-TIME PCR; LIGAMENT CELLS; QUANTITATIVE PCR; MINIMUM INFORMATION; HOUSEKEEPING GENES; UP-REGULATION; IN-VITRO; HYPOXIA; RNA; IDENTIFICATION;
Dewey Decimal Classification:	600 Technology > 610 Medical sciences Medicine
Status:	Published
Refereed:	Yes, this version has been refereed
Created at the University of Regensburg:	Yes
Item ID:	36212

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Abstract

Meaningful, reliable and valid mRNA expression analyses by real-time quantitative PCR (RT-qPCR) can only be achieved, if suitable reference genes are chosen for normalization and if appropriate RT-qPCR quality standards are met. Human periodontal ligament (hPDL) fibroblasts play a major mediating role in orthodontic tooth movement and periodontitis. Despite corresponding in-vitro gene expression ...

Abstract

Meaningful, reliable and valid mRNA expression analyses by real-time quantitative PCR (RT-qPCR) can only be achieved, if suitable reference genes are chosen for normalization and if appropriate RT-qPCR quality standards are met. Human periodontal ligament (hPDL) fibroblasts play a major mediating role in orthodontic tooth movement and periodontitis. Despite corresponding in-vitro gene expression studies being a focus of interest for many years, no information is available for hPDL fibroblasts on suitable reference genes, which are generally used in RT-qPCR experiments to normalize variability between samples. The aim of this study was to identify and validate suitable reference genes for normalization in untreated hPDL fibroblasts as well as experiments on orthodontic tooth movement or periodontitis (Aggregatibacter actinomycetemcomitans). We investigated the suitability of 13 candidate reference genes using four different algorithms (geNorm, NormFinder, comparative Delta C-q and BestKeeper) and ranked them according to their expression stability. Overall PPIB (peptidylprolyl isomerase A), TBP (TATA-box-binding protein) and RPL22 (ribosomal protein 22) were found to be most stably expressed with two genes in conjunction sufficient for reliable normalization. This study provides an accurate tool for quantitative gene expression analysis in hPDL fibroblasts according to the MIQE guidelines and shows that reference gene reliability is treatment-specific.

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Metadata last modified: 25 Nov 2020 20:53

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