Validation of Tuba1a as Appropriate Internal Control  for Normalization of Gene Expression Analysis during  Mouse Lung Development

Mehta, Aditi; Dobersch, Stephanie; Dammann, Reinhard; Bellusci, Saverio; Ilinskaya, О. N.; Braun, Thomas; Barreto, Guillermo

doi:10.3390/ijms16034492

Cited by 26 publications

(24 citation statements)

References 46 publications

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“…Ideally, the reference gene should have a stable expression in all cell types and in all experimental conditions. In mouse lung, the expression analysis of two tissue specific markers could also be up or downregulated depending on the reference gene chosen to normalize data (Mehta et al, 2015). Yet, many studies are published without proper validation and the consequences of choosing an inadequate reference gene could potentially lead to incorrect conclusions on gene expression analysis.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, the best reference genes alter according to the tissue analyzed, being different for all stomach tissues and stomach cancer cell lines (Rho et al, 2010). In mouse lung, the expression analysis of two tissue specific markers could also be up or downregulated depending on the reference gene chosen to normalize data (Mehta et al, 2015). Those observations exemplify the importance of adequate validation and critical analysis of published results.…”

Section: Discussionmentioning

confidence: 99%

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Cervical cancer stem‐like cells: systematic review and identification of reference genes for gene expression

Campos

Schultz

Mello

et al. 2017

Cell Biology International

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Cervical cancer is the fourth most common cancer affecting women worldwide. Among many factors, the presence of cancer stem cells, a subpopulation of cells inside the tumor, has been associated with a worse prognosis. Considering the importance of gene expression studies to understand the biology of cervical cancer stem cells (CCSC), this work identifies stable reference genes for cervical cancer cell lines SiHa, HeLa, and ME180 as well as their respective cancer stem-like cells. A literature review was performed to identify validated reference genes currently used to normalize RT-qPCR data in cervical cancer cell lines. Then, cell lines were cultured in regular monolayer or in a condition that favors tumor sphere formation. RT-qPCR was performed using five reference genes: ACTB, B2M, GAPDH, HPRT1, and TBP. Stability was assessed to validate the selected genes as suitable reference genes. The evaluation validated B2M, GAPDH, HPRT1, and TBP in these experimental conditions. Among them, GAPDH and TBP presented the lowest variability according to the analysis by Normfinder, Bestkeeper, and ΔC methods, being therefore the most adequate genes to normalize the combination of all samples. These results suggest that B2M, GAPDH, HPRT1, and TBP are suitable reference genes to normalize RT-qPCR data of established cervical cancer cell lines SiHa, HeLa, and ME180 as well as their derived cancer stem-like cells. Indeed, GAPDH and TBP seem to be the most convenient choices for studying gene expression in these cells in monolayers or spheres.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Cervical cancer stem‐like cells: systematic review and identification of reference genes for gene expression

Campos

Schultz

Mello

et al. 2017

Cell Biology International

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“…Quantitative real-time PCR reactions were performed using SYBR ® Green on the Step One plus Real-time PCR system (Applied Biosystem). Housekeeping genes Tuba1a and Gapdh were used to normalize gene expression [48]. Primer pairs used for gene expression analysis and Affymetrix microarray data analysis from WT and Hmga2 −/− mouse embryonic lung at E18.5 (GEO: GSE55340) are described in Supplementary information, Data S1.…”

Section: Rna Isolation Reverse Transcription Quantitative Pcr and mentioning

confidence: 99%

High mobility group protein-mediated transcription requires DNA damage marker γ-H2AX

et al. 2015

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The eukaryotic genome is organized into chromatins, the physiological template for DNA-dependent processes including replication, recombination, repair, and transcription. Chromatin-mediated transcription regulation involves DNA methylation, chromatin remodeling, and histone modifications. However, chromatin also contains non-histone chromatin-associated proteins, of which the high-mobility group (HMG) proteins are the most abundant. Although it is known that HMG proteins induce structural changes of chromatin, the processes underlying transcription regulation by HMG proteins are poorly understood. Here we decipher the molecular mechanism of transcription regulation mediated by the HMG AT-hook 2 protein (HMGA2). We combined proteomic, ChIP-seq, and transcriptome data to show that HMGA2-induced transcription requires phosphorylation of the histone variant H2AX at S139 (H2AXS139ph; γ-H2AX) mediated by the protein kinase ataxia telangiectasia mutated (ATM). Furthermore, we demonstrate the biological relevance of this mechanism within the context of TGFβ1 signaling. The interplay between HMGA2, ATM, and H2AX is a novel mechanism of transcription initiation. Our results link H2AXS139ph to transcription, assigning a new function for this DNA damage marker. Controlled chromatin opening during transcription may involve intermediates with DNA breaks that may require mechanisms that ensure the integrity of the genome.

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“…18S and GAPDH have been widely used as the reference for gene analysis in qRT-PCR [33]. However, our data showed that 18S was the least stable reference gene and GAPDH was not the best choice for gene analysis in HUVECs under H 2 O 2 treatment.…”

Section: Discussionmentioning

confidence: 78%

Identification of suitable reference genes for real-time quantitative PCR analysis of hydrogen peroxide-treated human umbilical vein endothelial cells

Diao

Zhao

et al. 2017

BMC Molecular Biol

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BackgroundOxidative stress can induce cell injury in vascular endothelial cells, which is the initial event in the development of atherosclerosis. Although quantitative real-time polymerase chain reaction (qRT-PCR) has been widely used in gene expression studies in oxidative stress injuries, using carefully validated reference genes has not received sufficient attention in related studies. The objective of this study, therefore, was to select a set of stably expressed reference genes for use in qRT-PCR normalization in oxidative stress injuries in human umbilical vein endothelial cells (HUVECs) induced by hydrogen peroxide (H2O2).ResultsUsing geNorm analysis, we found that five stably expressed reference genes were sufficient for normalization in qRT-PCR analysis in HUVECs treated with H2O2. Genes with the most stable expression according to geNorm were U6, TFRC, RPLP0, GAPDH, and ACTB, and according to NormFinder were ALAS1, TFRC, U6, GAPDH, and ACTB.ConclusionTaken together, our study demonstrated that the expression stability of reference genes may differ according to the statistical program used. U6, TFRC, RPLP0, GAPDH, and ACTB was the optimal set of reference genes for studies on gene expression performed by qRT-PCR assays in HUVECs under oxidative stress study.

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Validation of Tuba1a as Appropriate Internal Control for Normalization of Gene Expression Analysis during Mouse Lung Development

Cited by 26 publications

References 46 publications

Cervical cancer stem‐like cells: systematic review and identification of reference genes for gene expression

Cervical cancer stem‐like cells: systematic review and identification of reference genes for gene expression

High mobility group protein-mediated transcription requires DNA damage marker γ-H2AX

Identification of suitable reference genes for real-time quantitative PCR analysis of hydrogen peroxide-treated human umbilical vein endothelial cells

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