NDLI: Stable Reference Genes for qPCR Analysis in BM-MSCs Undergoing Osteogenic Differentiation within 3D Hyaluronan-Based Hydrogels

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Stable Reference Genes for qPCR Analysis in BM-MSCs Undergoing Osteogenic Differentiation within 3D Hyaluronan-Based Hydrogels

Content Provider	MDPI
Author	Hasler, Johannes Hatt, Luan Phelipe Stoddart, Martin James Armiento, Angela Rita
Copyright Year	2020
Description	Reverse transcription quantitative polymerase chain reaction (RT-qPCR) enables the monitoring of changes in cell phenotype via the high-throughput screening of numerous genes. RT-qPCR is a fundamental approach in numerous research fields, including biomaterials, yet little attention has been given to the potential impact of 3D versus monolayer (2D) cell culture and to the requirement for a constant validation of the multiple steps of gene expression analysis. The aim of this study is to use high-quality RNA to identify the most suitable reference genes for RT-qPCR analysis during the osteogenic differentiation of human bone marrow mesenchymal stem/stromal cells (BM-MSCs). BM-MSCs are cultured under osteogenic conditions for 28 days in 2D or within hyaluronic acid hydrogels (3D). RNA is subject to quality controls and is then used to identify the most stable reference genes using geNorm, NormFinder, and the ∆Cq method. The effect of the reverse transcriptase is investigated, as well as the expression of osteogenic-related markers. This study shows marked differences in the stability of reference genes between 2D (RPLP0/GAPDH) and 3D (OAZ1/PPIA) culture, suggesting that it is critical to choose appropriate reference genes for 3D osteogenic cell cultures. Thus, a thorough validation under specific experimental settings is essential to obtain meaningful gene expression results.
Starting Page	9195
e-ISSN	14220067
DOI	10.3390/ijms21239195
Journal	International Journal of Molecular Sciences
Issue Number	23
Volume Number	21
Language	English
Publisher	MDPI
Publisher Date	2020-12-02
Access Restriction	Open
Subject Keyword	International Journal of Molecular Sciences Cell Tissue Engineering Rt-qpcr Endogenous Control Rplp0 Oaz1 Ppia Biomaterials Osteogenesis Mesenchymal Stem Cells Tissue Engineering Hyaluronic Acid
Content Type	Text
Resource Type	Article

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