Validation of suitable house keeping genes for hypoxia-cultured human chondrocytes

Foldager, Casper Bindzus; Munir, Samir; Ulrik-Vinther, Michael; Søballe, Kjeld; Bünger, Cody; Lind, Martin

doi:10.1186/1471-2199-10-94

Cited by 102 publications

(89 citation statements)

References 42 publications

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“…However, for C. batrachus, ACTB and B2M, widely used RG internal controls (Lupberger et al 2002) were the least stable genes identified by all three analysis methods in examined tissues under normoxic and/or hypoxic conditions. These results were consistent with the earlier reports, which found ACTB to be highly regulated by treatments and have unstable expression in hypoxia-cultured human chondrocytes and lipopolysaccharide-stimulated human THP-1 and K562 cells (Cao et al 2012;Foldager et al 2009), whereas B2M was the least stable gene in human reticulocytes (Silver et al 2006). The present study on C. batrachus revealed novel candidate RGs (RPL30, TUB, 28S rRNA and ELF-1A) with a more stable expression in experimental contexts, in comparison to frequently used RGs (e.g.…”

Section: Discussionsupporting

confidence: 93%

Identification of candidate reference genes for quantitative expression analysis by real-time PCR for hypoxic stress in Indian catfish, Clarias batrachus (Linnaeus, 1758)

et al. 2014

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The selection of most appropriate reference gene(s) is a crucial step in studies quantifying gene expression by quantitative real-time PCR (qPCR). As a need reference gene(s) should be unaffected at transcription level by experimental conditions and/or tissue types. In this study, 11 candidate reference genes were tested in Clarias batrachus for their expression stability using the geNorm, NormFinder and BestKeeper statistical algorithms, and were compared in different tissues (brain, heart, liver, muscle, spleen and head kidney) and treatments (before and after hypoxia exposure). The results indicated that in brain, heart, liver, muscle, spleen and head kidney, respectively, 28S rRNA/TUB, 28S rRNA/TUB, RPL30/ 28S rRNA, RPL30/TUB, ELF-1A/28S rRNA and ELF-1A/TUB gene pairs were highly stable and were suitable as reference genes to study oxidative stress, while ACTB and B2M were the least stable genes in examined tissues under normoxic and/or hypoxic conditions. The observations suggested the consideration of tissue types and use of at least two reference genes, instead of one in accurate normalization of qPCR data.

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Section: Discussionsupporting

confidence: 93%

Identification of candidate reference genes for quantitative expression analysis by real-time PCR for hypoxic stress in Indian catfish, Clarias batrachus (Linnaeus, 1758)

et al. 2014

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“…In line with our results, Foldager et al found that TBP was the RG with the highest expression stability in 3D cultivated human chondrocytes. 29 In a study on human adipose-derived stem cells during hypoxic culture, osteogenic and chondrogenic differentiation, Fink et al identified TBP being among the most stable RGs. 28 The present data are in line with an earlier study of Kulkarni et al, in which TBP and HPRT1 were determined to be the most stable RGs in human cornea (46).…”

Section: Discussionmentioning

confidence: 99%

“…25,26 Commonly used RGs, for example, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta actin (ACTB), were observed to be less stable or regulated under certain culture conditions in MSCs, chondrocytes, and fibroblasts. [27][28][29][30] We recently showed that the combined use of DNA-damageinducible alpha (GADD45A), pumilio homolog 1 (PUM1), and large ribosomal protein P0 (RPLP0) significantly improved gene expression accuracy compared with classically used RGs, ACTB, and GAPDH in 2D osteogenically stimulated bone marrow-derived MSCs (BM-MSCs). 31 Numerous putative RGs have been reported for a wide variety of human cell lines and primary cells under different experimental conditions, almost exclusively under standardized 2D cultivation.…”

Section: Introductionmentioning

confidence: 99%

Identification of Stable Reference Genes for Gene Expression Analysis of Three-Dimensional Cultivated Human Bone Marrow-Derived Mesenchymal Stromal Cells for Bone Tissue Engineering

Rauh

Jacobi

Stiehler

2015

Tissue Engineering Part C: Methods

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The principles of tissue engineering (TE) are widely used for bone regeneration concepts. Three-dimensional (3D) cultivation of autologous human mesenchymal stromal cells (MSCs) on porous scaffolds is the basic prerequisite to generate newly formed bone tissue. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a specific and sensitive analytical tool for the measurement of mRNA-levels in cells or tissues.For an accurate quantification of gene expression levels, stably expressed reference genes (RGs) are essential to obtain reliable results. Since the 3D environment can affect a cell's morphology, proliferation, and gene expression profile compared with two-dimensional (2D) cultivation, there is a need to identify robust RGs for the quantification of gene expression. So far, this issue has not been adequately investigated. The aim of this study was to identify the most stably expressed RGs for gene expression analysis of 3D-cultivated human bone marrow-derived MSCs (BM-MSCs). For this, we analyzed the gene expression levels of n = 31 RGs in 3D-cultivated human BM-MSCs from six different donors compared with conventional 2D cultivation using qRT-PCR. MSCs isolated from bone marrow aspirates were cultivated on human cancellous bone cube scaffolds for 14 days. Osteogenic differentiation was assessed by cell-specific alkaline phosphatase (ALP) activity and expression of osteogenic marker genes. Expression levels of potential reference and target genes were quantified using commercially available TaqMan Ò assays. mRNA expression stability of RGs was determined by calculating the coefficient of variation (CV) and using the algorithms of geNorm and NormFinder. Using both algorithms, we identified TATA box binding protein (TBP), transferrin receptor (p90, CD71) (TFRC), and hypoxanthine phosphoribosyltransferase 1 (HPRT1) as the most stably expressed RGs in 3D-cultivated BMMSCs. Notably, genes that are routinely used as RGs, for example, beta actin (ACTB) and ribosomal protein L37a (RPL37A), were among the least stable genes. We recommend the combined use of TBP, TFRC, and HPRT1 for the accurate and robust normalization of qRT-PCR data of 3D-cultivated human BM-MSCs.

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“…To assess the level of contamination of the skin swab material by human DNA, we performed qPCR for a human housekeeping gene (GAPDH) which is present in all nucleated cells of human origin (10). The GAPDH qPCR was performed in parallel with the bacterial qPCRs.…”

Section: Validation Of Taqman Mgb Probesmentioning

confidence: 99%

Quantitation of Major Human Cutaneous Bacterial and Fungal Populations

Gao¹,

Perez-Perez²,

et al. 2010

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Because the human skin microbiota may play roles in the causation or modification of skin diseases, we sought to provide initial quantitative analysis from different cutaneous locations. We developed quantitative PCRs to enumerate the total bacterial and fungal populations, as well as the most common bacterial and fungal genera present in six locales, in eight healthy subjects. We used a set of primers and TaqMan MGB probes based on the bacterial 16S rRNA and fungal internally transcribed spacer region, as well as bacterial genus-specific probes for Propionibacterium, Corynebacterium, Streptococcus, and Staphylococcus and a fungal genus-specific probe for Malassezia. The extent of human DNA contamination of the specimen was determined by quantitating the human housekeeping GAPDH gene. The highest level of 16S rRNA copies of bacteria was present in the axilla (4.44 ؎ 0.18 log 10 copies/l [mean ؎ standard error of the mean]), with normalization based on GAPDH levels, but the other five locations were similar to one another (range, 2.48 to 2.89 log 10 copies/l). There was strong symmetry between the left and right sides. The four bacterial genera accounted for 31% to 59% of total bacteria, with the highest percent composition in the axilla and the lowest in the forearm. Streptococcus was the most common genus present on the forehead and behind the ear. Corynebacterium spp. were predominant in the axilla. Fungal levels were 1 to 2 log 10 lower than for bacteria, with Malassezia spp. accounting for the majority of fungal gene copies. These results provide the first quantitation of the site and host specificities of major bacterial and fungal populations in human skin and present simple methods for their assessment in studies of disease.Human skin harbors a diverse group of microorganisms that form complex communities and occupy specific niches and microenvironments (5,9,12,14,15). While most organisms colonizing the human body may be beneficial for health (4, 9, 18), some relationships with the host can change from commensal to pathogenic for reasons that are poorly understood (1). The global composition and presence of specific organisms also is relevant to the biological effects of the skin microbiota. The human cutaneous surface includes regions with diverse pH, temperature, moisture, and sebum content (14, 17), and skin structures such as hair follicles, sebaceous, eccrine, and apocrine glands comprise subhabitats that may be associated with their own unique microbiota (20).Until recently, our knowledge of the bacterial biota in human skin has been based mostly on cultivation studies, which are insufficient because many organisms cannot be cultured (6). The direct PCR amplification and sequencing of bacterial genes encoding the small subunit rRNA (16S rRNA) or variable region fragments thereof has been a powerful method to analyze the enormous variation in the human microbiome (8,24,30), showing important differences among a number of cutaneous sites (5, 9, 12, 15). However, analysis of ribosomal genes (16S rRNA ...

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Validation of suitable house keeping genes for hypoxia-cultured human chondrocytes

Cited by 102 publications

References 42 publications

Identification of candidate reference genes for quantitative expression analysis by real-time PCR for hypoxic stress in Indian catfish, Clarias batrachus (Linnaeus, 1758)

Identification of candidate reference genes for quantitative expression analysis by real-time PCR for hypoxic stress in Indian catfish, Clarias batrachus (Linnaeus, 1758)

Identification of Stable Reference Genes for Gene Expression Analysis of Three-Dimensional Cultivated Human Bone Marrow-Derived Mesenchymal Stromal Cells for Bone Tissue Engineering

Quantitation of Major Human Cutaneous Bacterial and Fungal Populations

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