Selection and validation of reference house­keeping genes in the J774A1 macrophage cell line for quantitative real-time PCR

Ferraz, Francielle Bonet; Fernández, Jorge Hernández

doi:10.4238/gmr.15017720

Cited by 10 publications

(6 citation statements)

References 36 publications

(59 reference statements)

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“…Quantitative PCR was conducted with SYBR Premix EX Taq under the conditions suggested by the manufacturer (Life Technologies). The quantity of respective genes was calculated by the comparative Ct method with glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) used as an internal reference (Ferraz & Fernandez, ).…”

Section: Methodsmentioning

confidence: 99%

Long‐term stability and characteristics of behavioral, biochemical, and molecular markers of three different rodent models for depression

et al. 2019

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Objective:The present study was designed to explore the long-term differences between three mouse models for depression. Method:In the present study, the unpredictable chronic mild stress (UCMS) model, the glucocorticoid/corticosterone model, and the olfactory bulbectomy model were compared at two, three, and five weeks after model induction. Behavioral testing performed included forced-swimming, tail suspension, open-field and elevated plusmaze tests. In addition, 5-hydroxytryptamine (5-HT) and dopamine levels, and mRNA and protein expressions related to 5-HT synthesis, transport, and signaling were analyzed in the hippocampus of tested animals. Results:Our results revealed that each model demonstrated a specific profile of markers, whereas the stability of them differed over testing time. Conclusions:Each model provided a unique set of advantages that can be considered depending on the context and aims of each study. Among the three models, the UCMS model was mostly stable and appeared to the best model for testing long-term depression-like state. K E Y W O R D S 5-HT, corticosterone, depression model, glucocorticoid, olfactory bulbectomy, UCMS How to cite this article: Zhu H, Tao Y, Wang T, et al. Long-term stability and characteristics of behavioral, biochemical, and molecular markers of three different rodent models for depression. Brain Behav. 2020;10:e01508. https ://doi.

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Section: Methodsmentioning

confidence: 99%

Long‐term stability and characteristics of behavioral, biochemical, and molecular markers of three different rodent models for depression

et al. 2019

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“…Only few studies reported results of gene expression in monocytes/macrophages in sepsis experimental approaches ( Table 1). The authors mostly performed in vitro studies using mice bone marrow-derived monocytes/macrophages [18,22], J774A1 murine macrophage cell line [23], monocytes from healthy donors stimulated with LPS [24] and monocytes from patients with relapsing-remitting multiple sclerosis [25]. It is notable the lack of study in monocytes/macrophages from septic patients.…”

Section: Discussionmentioning

confidence: 99%

Reference Genes for Quantitative qPCR Analyses in Monocytes of Septic Patients

Rb¹,

Souza‐Siqueira²,

Ln³

et al. 2020

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Abstract Background: Monocytes and macrophages are essential components of the innate and adaptive immune responses and play a critical role in sepsis. Sepsis is a life-threatening organ dysfunction associated with an unregulated host response to infection. About 20 million people develop sepsis annually, and up to 50% die. There is a lack of studies regarding human monocytes and sepsis. This study aimed to determine the most stable internal gene (s) to investigate gene expression in monocytes/macrophages of septic patients.Methods: The expression stability of fifteen commonly used reference genes was analyzed by determining the comparative threshold cycle (Ct) values, using the BestKeeper, GeNorm, and NormFinder algorithms.Results: BestKeeper analysis revealed that the syntaxin 5 (STX5A) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) genes were highly stable. GeNorm pointed out STX5A and phosphoglycerate kinase 1 (PGK1) as the most suitable combination whereas through NormFinder glyceraldehyde 3- phosphate dehydrogenase (GAPDH) and 14-3-3 zeta/delta protein (YWHAZ) was the most stable combination. All programs analysis discarded the use of heterogeneous nuclear ribonucleoprotein A/B (HNRNPAB). GeNorm and NormFinder indicated actin-beta (ACTB) as the least stable gene.Conclusions: The combined data indicated that STX5A, PGK1, GAPDH, and HPRT1 are highly suitable reference genes for qPCR analysis of septic patients monocytes. In the case of choosing one single reference gene, the results point out to STX5A (first place by GeNorm and BestKeeper and third place by NormFinder). This study is the first report on reference genes in monocytes/macrophages from septic patients.

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“…qPCR is one of the most sensitive method to evaluate the expression level of genes of interest under different experimental conditions, and thus in order to obtain reliable results it is crucial to select the suitable reference genes [ 26 , 29 – 34 ].…”

Section: Discussionmentioning

confidence: 99%

Identification of reference genes for quantitative PCR during C3H10T1/2 chondrogenic differentiation

et al. 2019

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C3H10T1/2, a mouse mesenchymal stem cell line, is a well-known in vitro model of chondrogenesis that can be easily employed to recapitulate some of the mechanisms intervening in this process. Moreover, these cells can be used to validate the effect of candidate molecules identified by high throughput screening approaches applied to the development of targeted therapy for human disorders in which chondrogenic differentiation may be involved, as in conditions characterized by heterotopic endochondral bone formation. Chondrogenic differentiation of C3H10T1/2 cells can be monitored by applying quantitative polymerase chain reaction (qPCR), one of the most sensitive methods that allows detection of small dynamic changes in gene expression between samples obtained under different experimental conditions. In this work, we have used qPCR to monitor the expression of specific markers during chondrogenic differentiation of C3H10T1/2 cells in micromass cultures. Then we have applied the geNorm approach to identify the most stable reference genes suitable to get a robust normalization of the obtained expression data. Among 12 candidate reference genes ( Ap3d1, Csnk2a2, Cdc40, Fbxw2, Fbxo38, Htatsf1, Mon2, Pak1ip1, Zfp91, 18S, ActB, GAPDH ) we identified Mon2 and Ap3d1 as the most stable ones during chondrogenesis. ActB, GAPDH and 18S , the most commonly used in the literature, resulted to have an expression level too high compared to the differentiation markers ( Sox9, Collagen type 2a1, Collagen type 10a1 and Collagen type 1a1 ), therefore are actually less recommended for these experimental conditions. In conclusion, we identified nine reference genes that can be equally used to obtain a robust normalization of the gene expression variation during the C3H10T1/2 chondrogenic differentiation. Electronic supplementary material The online version of this article (10.1007/s11033-019-04713-x) contains supplementary material, which is available to authorized users.

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Selection and validation of reference housekeeping genes in the J774A1 macrophage cell line for quantitative real-time PCR

Cited by 10 publications

References 36 publications

Long‐term stability and characteristics of behavioral, biochemical, and molecular markers of three different rodent models for depression

Long‐term stability and characteristics of behavioral, biochemical, and molecular markers of three different rodent models for depression

Reference Genes for Quantitative qPCR Analyses in Monocytes of Septic Patients

Identification of reference genes for quantitative PCR during C3H10T1/2 chondrogenic differentiation

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Selection and validation of reference house­keeping genes in the J774A1 macrophage cell line for quantitative real-time PCR

Cited by 10 publications

References 36 publications

Long‐term stability and characteristics of behavioral, biochemical, and molecular markers of three different rodent models for depression

Long‐term stability and characteristics of behavioral, biochemical, and molecular markers of three different rodent models for depression

Reference Genes for Quantitative qPCR Analyses in Monocytes of Septic Patients

Identification of reference genes for quantitative PCR during C3H10T1/2 chondrogenic differentiation

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Selection and validation of reference housekeeping genes in the J774A1 macrophage cell line for quantitative real-time PCR