RT-qPCR analyses on the osteogenic differentiation from human iPS cells: an investigation of reference genes

Okamura, Kensuke; Inagaki, Yusuke; Matsui, Takeshi K.; Matsubayashi, Masaya; Komeda, Tomoya; Ogawa, Makoto; Mori, Eiichiro; Tanaka, Yasuhito

doi:10.1038/s41598-020-68752-2

Cited by 18 publications

(23 citation statements)

References 32 publications

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“…Phosphoglycerate kinase 1 (PGK1) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are glycolytic enzyme involved in several metabolic pathways that are essential for cell growth and proliferation. The expression of these genes has been shown to differ in different tissue types and environment conditions 54 , 55 because of its functions in transcriptional and posttranscriptional gene regulation, intracellular membrane trafficking, DNA replication and reparation 56 , 57 .…”

Section: Resultsmentioning

confidence: 99%

Evaluation of candidate reference genes for quantitative real-time PCR normalization in blood from red deer developing antlers

Broggini

Abril

Carranza

et al. 2022

Sci Rep

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Sexual selection favors male traits that increase their ability to monopolize the breeding access to several females. Deer antlers are cranial appendages that regenerate annually in males. Throughout life, the phenology of antler growth advances and antler mass increases until the stag reaches, between 8 and 10 years old, maximum body mass and highest reproductive success. The molecular mechanisms of antler development are of great interest in both evolutionary and regenerative medicine studies. To minimize errors in the assessment of gene expression levels by qRT-PCR, we analyzed the stability of a panel of eight candidate reference genes and concluded that qRT-PCR normalization to three stable genes is strongly convenient in experiments performed in red deer antler blood. To validate our proposal, we compared the expression level of three genes linked to red deer antler growth (ANXA2, APOD and TPM1) in fifteen male red deer classified as young (up to 4 years old) and adults (4–6 years old). Our data confirms that B2M, ACTB and RPLP0 are valuable reference genes for future gene expression studies in red deer antler blood, which would provide increased insight into the effects of intrinsic factors that determine antler development in red deer.

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Section: Resultsmentioning

confidence: 99%

Evaluation of candidate reference genes for quantitative real-time PCR normalization in blood from red deer developing antlers

Broggini

Abril

Carranza

et al. 2022

Sci Rep

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“…The specimens were subjected to PBS washing three times after culturing the specimens for 14 days, and then they were put off in cold TRIzol Reagent (1 ml). The total RNA of each sample was extracted and again put off in RNase-free water (50 μL) using the standard TRIzol protocol (Okamura et al, 2020). Following the protocol, the cDNA was created and stored at -20 °C until further analysis.…”

Section: Osteogenic Differentiation By Gene Expression Studiesmentioning

confidence: 99%

Hydroxyapatite-collagen- carboxylic carbon quantum dot composite loaded with chrysin supported the proliferation and differentiation of human bone marrow derived mesenchymal stem cells

Zhou

Chen²,

Mickymaray³

et al. 2022

Front. Mater.

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Developing a bioactive scaffold with biocompatible material is a substantial approach to bone regeneration and functional healing. Hydroxyapatite (HAP) is the main component in bone formation as an inorganic component and regeneration due to its osteoconductive properties. In this study, we prepared a scaffold material composed of HAP and collagen (COL) cross-linked via carboxylic carbon quantum dots (CCQD) with a chrysin (CRN) molecule. CRN is a flavonoid that has been shown to encourage the bone development of bone marrow-derived mesenchymal stem cells. It is loaded for enhancing bone regeneration and HAP’s growth ability. XRD, FT-IR, SEM, and TEM analysis have characterized the prepared composites for their crystalline nature, functional behavior, and morphological evaluations. The HAP has retained its original crystalline lattice confirmed from XRD analysis in the prepared composites. The addition of CRN molecule has decreased the length of HAP rods from ∼932 nm to ∼459 nm, as confirmed by TEM images. The increased particle sizes have been observed for the prepared composites. It reaches the maximum at 938.0 nm for the final HAP/COL/CCQD/CRN composite, which was confirmed by particle size analysis. The in-vitro CRN release behavior shows that the CRN molecule has controlled release up to 23% for 48 h. The biocompatibility of prepared material was investigated and confirmed on human bone marrow-derived mesenchymal stem cells (hBMSCs). This examination has proven that the prepared material is good for bone cell regeneration. The material may apply for bone regeneration applications after in-vivo and clinical investigations.

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“…Additionally, various methodology articles have made further efforts to ensure the stability and optimal quantity of reference genes to obtain accurately reproducible data, providing considerable impetus towards perfecting RT-qPCR [19][20][21][22]. Subsequent bone and cartilage bioengineering studies have suggested that the stability and normalization quantity of reference genes should not only be determined by cell or tissue type but should also be re-optimized for experiments under different processing conditions [23,24], which has provided an extra step in perfecting the understanding of the MIQE guidelines.…”

Section: Introductionmentioning

confidence: 99%

Optimizations for identifying reference genes in bone and cartilage bioengineering

et al. 2021

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Background Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) remains one of the best-established techniques to assess gene expression patterns. However, appropriate reference gene(s) selection remains a critical and challenging subject in which inappropriate reference gene selction can distort results leading to false interpretations. To date, mixed opinions still exist in how to choose the most optimal reference gene sets in accodrance to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guideline. Therefore, the purpose of this study was to investigate which schemes were the most feasible for the identification of reference genes in a bone and cartilage bioengineering experimental setting. In this study, rat bone mesenchymal stem cells (rBMSCs), skeletal muscle tissue and adipose tissue were utilized, undergoing either chondrogenic or osteogenic induction, to investigate the optimal reference gene set identification scheme that would subsequently ensure stable and accurate interpretation of gene expression in bone and cartilage bioengineering. Results The stability and pairwise variance of eight candidate reference genes were analyzed using geNorm. The V0.15- vs. Vmin-based normalization scheme in rBMSCs had no significant effect on the eventual normalization of target genes. In terms of the muscle tissue, the results of the correlation of NF values between the V0.15 and Vmin schemes and the variance of target genes expression levels generated by these two schemes showed that different schemes do indeed have a significant effect on the eventual normalization of target genes. Three selection schemes were adopted in terms of the adipose tissue, including the three optimal reference genes (Opt3), V0.20 and Vmin schemes, and the analysis of NF values with eventual normalization of target genes showed that the different selection schemes also have a significant effect on the eventual normalization of target genes. Conclusions Based on these results, the proposed cut-off value of Vn/n + 1 under 0.15, according to the geNorm algorithm, should be considered with caution. For cell only experiments, at least rBMSCs, a Vn/n + 1 under 0.15 is sufficient in RT-qPCR studies. However, when using certain tissue types such as skeletal muscle and adipose tissue the minimum Vn/n + 1 should be used instead as this provides a far superior mode of generating accurate gene expression results. We thus recommended that when the stability and variation of a candidate reference genes in a specific study is unclear the minimum Vn/n + 1 should always be used as this ensures the best and most accurate gene expression value is achieved during RT-qPCR assays.

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RT-qPCR analyses on the osteogenic differentiation from human iPS cells: an investigation of reference genes

Cited by 18 publications

References 32 publications

Evaluation of candidate reference genes for quantitative real-time PCR normalization in blood from red deer developing antlers

Evaluation of candidate reference genes for quantitative real-time PCR normalization in blood from red deer developing antlers

Hydroxyapatite-collagen- carboxylic carbon quantum dot composite loaded with chrysin supported the proliferation and differentiation of human bone marrow derived mesenchymal stem cells

Optimizations for identifying reference genes in bone and cartilage bioengineering

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