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

Abstract

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.

Identifying the best reference gene for RT-qPCR analyses of the three-dimensional osteogenic differentiation of human-induced pluripotent stem cells

“…Therefore, RPS18 should not be considered an appropriate reference gene in future studies 19,20 . Our results are consistent with those of Hasler et al 3 , who reported that despite this gene showing stable expression in 3D culture, it may not be a suitable reference gene. We ranked the most stable reference genes in the following order: TBP > HPRT1 > YWHAZ.…”

“…Our results are consistent with their ndings; we found that TBP and HPRT1 showed stable expression. Hasler et al 3 veri ed the optimal reference genes from eight endogenous control genes in 3D osteogenic differentiation using BM-MSCs using geNorm, NormFinder, and ΔCt method. They reported that PPIA, OAZ1, and GUSB were the most suitable reference genes for normalization, with TBP ranking fth.…”

“…The reference gene chosen for normalization should exhibit stable expression throughout experiments and across different groups. However, inappropriate normalization remains one of the pitfalls of qPCR; further, this issue is often underreported in gene expression studies 3 .…”

“…Therefore, in tissue engineering, it is necessary to replicate the 3D microenvironment using natural or synthetic materials. Furthermore, 3D culture can in uence cell morphology, proliferation, and gene expression compared to 2D culture, necessitating the identi cation of robust reference genes for quantitative gene expression analyses 3 . The objective of this study was to identify the most stably expressed reference genes for gene expression analysis during osteogenic differentiation induction experiments, using hiPSCs cultured in a 3D environment.…”

Assessment of Reference Genes Stability in Cortical Bone of Obese and Diabetic Mice

“…Bone tissue is comprised of bone cells (predominantly osteocytes) and abundant bone matrix, and it is difficult to obtain high-quality RNA for identification of stable reference genes. 37 Some investigations have validated the stability of reference genes in 1086 osteogenic differentiation during various treatments, [38][39][40][41] but only a few have focused on bone tissue, 20,42 particularly in the context of metabolic disorders. Several advancements in methods for studying metabolic disorders have been made recently.…”