Reference genes for gene expression studies in the mouse heart

Ruiz‐Villalba, Adrián; Mattiotti, Andrea; Gunst, Quinn D.; Cano-Ballesteros, Sara; Hoff, Maurice J.B. van den; Ruijter, Jan M.

doi:10.1038/s41598-017-00043-9

Cited by 53 publications

(31 citation statements)

References 19 publications

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“…On the other hand, our analysis has revealed that the expression pattern of the actin gene (Actb), often considered as a reliable reference gene in other studies 32 , steadily decreased throughout the reprogramming. As a result, this gene was consistently ranked as one of the most unstable genes across different statistical methods.…”

Section: Discussionmentioning

confidence: 78%

Validation of Common Housekeeping Genes as Reference for qPCR Gene Expression Analysis During iPS Reprogramming Process

Panina

Germond

Masui

et al. 2018

Sci Rep

101

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Induced pluripotent stem cell (iPS) reprogramming allows to turn a differentiated somatic cell into a pluripotent cell. This process is accompanied by many changes in fundamental cell properties, such as energy production, cell-to-cell interactions, cytoskeletal organization, and others. Real-time quantitative polymerase chain reaction (RT-qPCR) can be used as a quantitative method of gene expression analysis to investigate iPS reprogramming but it requires a validation of reference genes for the accurate assessment of target genes’ expression. Currently, studies evaluating the performance of reference genes during iPS reprogramming are lacking. In this study we analysed the stability of 12 housekeeping genes during 20 days of iPS reprogramming of murine cells based on statistical analyses of RT-qPCR data using five different statistical algorithms. This study reports strong variations in housekeeping gene stability during the reprogramming process. Most stable genes were Atp5f1, Pgk1 and Gapdh, while the least stable genes were Rps18, Hprt, Tbp and Actb. The results were validated by a proof-of-point qPCR experiment with pluripotent markers Nanog, Rex1 and Oct4 normalized to the best and the worst reference gene identified by the analyses. Overall, this study and its implications are particularly relevant to investigations on the cell-state and pluripotency in iPS reprogramming.

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Section: Discussionmentioning

confidence: 78%

Validation of Common Housekeeping Genes as Reference for qPCR Gene Expression Analysis During iPS Reprogramming Process

Panina

Germond

Masui

et al. 2018

Sci Rep

101

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“…30 The expression patterns of control genes are presented in Supplemental Figure S3B. Expression of Actb, Rpl4, Rpl32, Tbp, Oaz1 and Pgk1 did not change significantly, whereas several other genes either generally used or recommended as control genes 31 , such as Gapdh, were up-or downregulated (q < 0.01 and fold change > 1.5) in at least one time-point comparison. Differential expression analysis of all protein-coding genes is available in Supplemental Dataset S1.…”

Section: Transcriptomicsmentioning

confidence: 99%

Molecular atlas of postnatal mouse heart development

Talman

Teppo

Pöhö

et al. 2018

Preprint

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Rationale: Mammals lose the ability to regenerate their hearts within one week after birth. During this regenerative window, cardiac energy metabolism shifts from glycolysis to fatty acid oxidation, and recent evidence suggests that metabolism may participate in controlling cardiomyocyte cell cycle. However, the molecular mechanisms mediating the loss of postnatal cardiac regeneration are not fully understood.Objective: This study aims at providing an integrated resource of mRNA, protein and metabolite changes in the neonatal heart to identify metabolism-related mechanisms associated with the postnatal loss of regenerative capacity. Methods and Results: Mouse ventricular tissue samples taken on postnatal days 1, 4, 9 and 23 (P01, P04, P09 and P23, respectively) were analyzed with RNA sequencing (RNAseq) and global proteomics and metabolomics. Differential expression was observed for 8547 mRNAs and for 1199 of the 2285 quantified proteins. Furthermore, 151 metabolites with significant changes were identified. Gene ontology analysis, KEGG pathway analysis and fuzzy c-means clustering were used to identify biological processes and metabolic pathways either up-or downregulated on all three levels. Among these were branched chain amino acid degradation (upregulated at P23) and production of free saturated and monounsaturated medium-to long-chain fatty acids (upregulated at P04 and P09; downregulated at P23). Moreover, the HMG-CoA synthase (HMGCS)-mediated mevalonate pathway and ketogenesis were transiently activated. Pharmacological inhibition of HMGCS in primary neonatal rat ventricular cardiomyocytes reduced the percentage of BrdU+ cardiomyocytes, providing evidence that the mevalonate and ketogenesis routes may participate in regulating cardiomyocyte cell cycle. Conclusions: This is the first systems-level resource combining data from genome-wide transcriptomics with global quantitative proteomics and untargeted metabolomics analyses of the mouse heart throughout the early postnatal period. This integrated multi-level data of molecular changes associated with the loss of cardiac regeneration may open up new possibilities for the development of regenerative therapies.

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“…qRT-PCR was performed using SYBR Green (Roche) on a Lightcycler 480 system II (Roche). In case of multi-tissue qPCRs, the geometric mean of multiple reference genes was used [ 32 ]. Analysis of qRT-PCR data was done using LinRegPCR analysis software [ 33 ].…”

Section: Methodsmentioning

confidence: 99%

The RNA-binding protein Rbm38 is dispensable during pressure overload-induced cardiac remodeling in mice

et al. 2017

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The importance of tightly controlled alternative pre-mRNA splicing in the heart is emerging. The RNA binding protein Rbm24 has recently been identified as a pivotal cardiac splice factor, which governs sarcomerogenesis in the heart by controlling the expression of alternative protein isoforms. Rbm38, a homolog of Rbm24, has also been implicated in RNA processes such as RNA splicing, RNA stability and RNA translation, but its function in the heart is currently unknown. Here, we investigated the role of Rbm38 in the healthy and diseased adult mouse heart. In contrast to the heart- and skeletal muscle-enriched protein Rbm24, Rbm38 appears to be more broadly expressed. We generated somatic Rbm38 -/- mice and show that global loss of Rbm38 results in hematopoietic defects. Specifically, Rbm38 -/- mice were anemic and displayed enlarged spleens with extramedullary hematopoiesis, as has been shown earlier. The hearts of Rbm38 -/- mice were mildly hypertrophic, but cardiac function was not affected. Furthermore, Rbm38 deficiency did not affect cardiac remodeling (i.e. hypertrophy, LV dilation and fibrosis) or performance (i.e. fractional shortening) after pressure-overload induced by transverse aorta constriction. To further investigate molecular consequences of Rbm38 deficiency, we examined previously identified RNA stability, splicing, and translational targets of Rbm38. We found that stability targets p21 and HuR, splicing targets Mef2d and Fgfr2, and translation target p53 were not altered, suggesting that these Rbm38 targets are tissue-specific or that Rbm38 deficiency may be counteracted by a redundancy mechanism. In this regard, we found a trend towards increased Rbm24 protein expression in Rbm38 -/- hearts. Overall, we conclude that Rbm38 is critical in hematopoiesis, but does not play a critical role in the healthy and diseased heart.

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Reference genes for gene expression studies in the mouse heart

Cited by 53 publications

References 19 publications

Validation of Common Housekeeping Genes as Reference for qPCR Gene Expression Analysis During iPS Reprogramming Process

Validation of Common Housekeeping Genes as Reference for qPCR Gene Expression Analysis During iPS Reprogramming Process

Molecular atlas of postnatal mouse heart development

The RNA-binding protein Rbm38 is dispensable during pressure overload-induced cardiac remodeling in mice

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