scholarly journals Identification of Appropriate Reference Genes for qRT-PCR Analysis of Heat-Stressed Mammary Epithelial Cells in Riverine Buffaloes (Bubalus bubalis)

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Neha Kapila ◽  
Amit Kishore ◽  
Monika Sodhi ◽  
Ankita Sharma ◽  
Pawan Kumar ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Reference Genes ◽  
Mammary Epithelial Cells ◽  
Bubalus Bubalis ◽  
Mammary Epithelial ◽  
Pcr Analysis ◽  
Functional Classes ◽  
Gene Expression Studies ◽  
The Stability ◽  
Suitable Reference

Gene expression studies require appropriate normalization methods for proper evaluation of reference genes. To date, not many studies have been reported on the identification of suitable reference genes in buffaloes. The present study was undertaken to determine the panel of suitable reference genes in heat-stressed buffalo mammary epithelial cells (MECs). Briefly, MEC culture from buffalo mammary gland was exposed to 42 °C for one hour and subsequently allowed to recover at 37 °C for different time intervals (from 30 m to 48 h). Three different algorithms, geNorm, NormFinder, and BestKeeper softwares, were used to evaluate the stability of 16 potential reference genes from different functional classes. Our data identified RPL4, EEF1A1, and RPS23 genes to be the most appropriate reference genes that could be utilized for normalization of qPCR data in heat-stressed buffalo MECs.

scholarly journals Review of: Metalloproteinase axes increase β-catenin signaling in primary mouse mammary epithelial cells lacking TIMP3

2007 ◽  
Vol 10 (8) ◽  
Author(s):  
D. S. Salomon
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Target Genes ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Specific Effects ◽  
Primary Mouse ◽  
Ductal Elongation

Citation of original article:C. V. Hojilla, I. Kim, Z. Kassiri, J. E. Fat, H. Fang, R. Khokha. Journal of Cell Science 2007; 120(6): 1050–1060.Abstract of the original article:Multiple cancers exhibit mutations in β-catenin that lead to increased stability, altered localization or amplified activity. β-Catenin is situated at the junction between the cadherin-mediated cell adhesion and Wnt signaling pathways, and TIMP3 functions to alter β-catenin signaling. Here we demonstrate that primary mouse embryonic fibroblasts (MEFs) and mammary epithelial cells (MECs) deficient in Timp3 have increased β-catenin signaling. Functionally, the loss of TIMP3 exerted cell-type-specific effects, with Timp3−/− MEFs being more sensitive and Timp3−/− MECs more resistant to EGTA-induced cell detachment than the wild type. Timp3−/− MECs had higher dephosphorylated β-catenin levels and increased β-catenin transcriptional activity as measured by TCF/LEF-responsive reporter assays. Real-time PCR analysis of β-catenin target genes in MEFs and MECs showed no alteration in Myc, decreased Ccnd1 (cyclin D1) and increased Mmp7 mRNA levels upon loss of TIMP3, with the latter occurring only in epithelial cells. Recombinant TIMP3 and synthetic metalloproteinase inhibitors reverted the increase in dephosphorylated β-catenin, decrease in Ccnd1 gene expression and increase in Mmp7 gene expression. Physiologically, Timp3−/− mammary glands displayed accelerated mammary ductal elongation during pubertal morphogenesis. Gain-of-function studies using slow-release TIMP-containing pellets revealed distinct effects of individual TIMPs on ductal morphogenesis. Recombinant TIMP1, TIMP3 and TIMP4 inhibited ductal elongation whereas TIMP2 promoted this process.

scholarly journals Leptin Expression in Human Mammary Epithelial Cells and Breast Milk

1998 ◽  
Vol 83 (5) ◽  
pp. 1810-1810 ◽  
Author(s):  
Susan M. Smith-Kirwin ◽  
Darlise M. O’Connor ◽  
Jennifer Johnston ◽  
Elizabeth de Lancy ◽  
Sandra G. Hassink ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Breast Milk ◽  
Epithelial Cells ◽  
Milk Fat ◽  
Mammary Epithelial Cells ◽  
Skim Milk ◽  
Mammary Epithelial ◽  
Pcr Analysis ◽  
Human Mammary Epithelial Cells ◽  
Human Mammary Epithelial

Leptin has recently been shown to be produced by the human placenta and potentially plays a role in fetal and neonatal growth. Many functions of the placenta are replaced by the mammary gland in terms of providing critical growth factors for the newborn. In this study, we show that leptin is produced by human mammary epithelial cells as revealed by RT/PCR analysis of total RNA from mammary gland and immunohistochemical staining of breast tissue, cultured mammary epithelial cells, and secretory epithelial cells present in human milk. We also verify that immunoreactive leptin is present in whole milk at 30- to 150-fold higher concentrations than skim milk. We propose that leptin is secreted by mammary epithelial cells in milk fat globules, which partition into the lipid portion of breast milk.

scholarly journals The Effects of Triazine Herbicides on Critical Peroxide Metabolism Gene Expression in MCF-7 and MCF-10A Cells

2020 ◽  
pp. 71-82
Author(s):  
Stephen J. DeMartini ◽  
Nicole B. DeHart ◽  
Jennifer R. Schroeder
Keyword(s):  
Gene Expression ◽  
Hydrogen Peroxide ◽  
Mammary Epithelial Cells ◽  
Human Mammary Epithelial Cell ◽  
Mammary Epithelial ◽  
Pcr Analysis ◽  
Triazine Herbicides ◽  
Human Mammary Epithelial ◽  
The Stability ◽  
Mcf 7

Aim: To identify the oxidative stress impacts of chloro-s-triazine herbicides on human mammary epithelial cell lines. Study Design: MCF-7 mammary epithelial carcinoma and MCF-10A mammary epithelial cells were treated with levels of three triazine herbicides in concentrations flanking the US FDA safe levels. Place and Duration of Study: Department of Biology, Millikin University, in January 2015 through December 2015 and January 2019 through May 2020. Methodology: We examined the oxidative effects of two triazine herbicides, atrazine and simazine, on estrogen-dependent MCF-7 mammary epithelial carcinoma cells using three different bioluminescent assay techniques. We then utilized real time PCR to analyze gene expression through RT-PCR analysis, in both MCF-7 cells and a non-cancerous cell line, MCF-10A, for both of these triazine herbicides plus the related cyanazine. Results: At all concentrations of atrazine and simazine, no statistical differences were found in the levels of oxidized glutathione or total oxidized and reduced nicotinamide adenine dinucleotides phosphates. In stark contrast, levels of hydrogen peroxide were found to be statistically different from the control at all concentrations of atrazine and simazine tested. Using an Analysis of Variance (ANOVA) we determined that within the enzymatic portion of the hydrogen peroxide pathway there were statistically significant differences in the expression of Peroxiredoxin 1 (PRDX1), Sulfiredoxin (SRXN1), and Thioredoxin (TXN). Conclusion: Exposure to triazines alters the hydrogen peroxide pathway, which in turn can greatly affect the stability of the cell milieu.

scholarly journals Determination of reliable reference genes for gene expression studies in Chinese chive (Allium tuberosum) based on the transcriptome profiling

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jing Tong ◽  
Manman Hu ◽  
Beibei Han ◽  
Yanhai Ji ◽  
Baoju Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Transcriptome Profiling ◽  
Pcr Analysis ◽  
Allium Tuberosum ◽  
Qrt Pcr ◽  
Complex Processes ◽  
Chinese Chive ◽  
Gene Expression Studies ◽  
The Stability

AbstractChinese chive (Allium tuberosum) is widely cultivated around the world for its unique flavor, nutrient, and medicinal values, yet its molecular mechanism on flavor formation and other metabolic pathways remains intangible. The elucidation of these complex processes begins with investigating the expression of the genes of interest, however the appropriate reference genes (RGs) for normalizing the gene expression are still unavailable in A. tuberosum. To fill this lacuna, transcriptome-wide screening was undertaken to identify the most stable genes according to the analysis of their FPKM values. The expression stability of the RGs was further evaluated using geNorm, NormFinder, BestKeeper, and RefFinder algorithms. The comprehensive analysis showed that GLY1 and SKP1, instead of two traditionally used RGs (eIF1α and ACT2), were the most stable genes across diverse A. tuberosum tissues, indicating the necessity to carefully validate the stability of RGs prior to their use for normalizations. As indicated by geNorm, the normalizations with at least two RGs could give more accurate results. qRT-PCR experiments were conducted with randomly selected genes, demonstrating that normalization with a combination of GLY1 and SKP1 resulted in reliable normalization results. Our finding represents the first attempt toward establishing a standardized qRT-PCR analysis in this economically important vegetable.

Screening of reference genes using real-time quantitative PCR for gene expression studies inNeoseiulus barkeriHughes (Acari: Phytoseiidae)

2018 ◽  
Vol 109 (4) ◽  
pp. 443-452 ◽  
Author(s):  
C. Wang ◽  
J. Yang ◽  
Q. Pan ◽  
S. Yu ◽  
R. Luo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Genes ◽  
Developmental Stages ◽  
Quantitative Polymerase Chain Reaction ◽  
Real Time Quantitative Pcr ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
The Stability ◽  
Suitable Reference

AbstractA stable reference gene is a key prerequisite for accurate assessment of gene expression. At present, the real-time reverse transcriptase quantitative polymerase chain reaction has been widely used in the analysis of gene expression in a variety of organisms.Neoseiulus barkeriHughes (Acari: Phytoseiidae) is a major predator of mites on many important economically crops. Until now, however, there are no reports evaluating the stability of reference genes in this species. In view of this, we used GeNorm, NormFinder, BestKeeper, and RefFinder software tools to evaluate the expression stability of 11 candidate reference genes in developmental stages and under various abiotic stresses. According to our results, β-ACTandHsp40were the top two stable reference genes in developmental stages. TheHsp60andHsp90were the most stable reference genes in various acaricides stress. For alterations in temperature,Hsp40and α-TUBwere the most suitable reference genes. About UV stress,EF1α and α-TUBwere the best choice, and for the different prey stress, β-ACTand α-TUBwere best suited. In normal conditions, the β-ACT and α-TUB were the two of the highest stable reference genes to respond to all kinds of stresses. The current study provided a valuable foundation for the further analysis of gene expression inN. barkeri.

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