Identification of suitable housekeeping genes for expression analysis in mammary epithelial cells of buffalo (Bubalus bubalis) during lactation cycle

Owing to the difficulty in obtaining mammary gland tissue from lactating animals, it is difficult to test the expression levels of genes in mammary gland. The aim of the current study was to identify if milk fat globule (MFG) in buffalo milk was an alternative to mammary gland (MG) and milk somatic cell (MSC) for gene expression analysis. Six buffalos in late lactation were selected to collect MFG and MSC, and then MG was obtained by surgery. MFG was stained with acridine orange to successfully visualise RNA and several cytoplasmic crescents in MFG. The total RNA in MFG was successfully isolated and the integrity was assessed by agarose gel electrophoresis. We analysed the cellular components in MFG, MG and MSC through testing the expression of cell-specific genes by qRT-PCR. The results showed that adipocyte-specific gene (AdipoQ) and leucocyte-specific genes (CD43, CSF1 and IL1α) in MFG were not detected, whereas epithelial cell marker genes (Keratin 8 and Keratin 18) in MFG were higher than in MSC and lower than in MG, fibroblast marker gene (vimentin) in MFG was significantly lower than in MG and MSC, milk protein genes (LALBA, BLG and CSN2) and milk fat synthesis-related genes (ACC, BTN1A1, FABP3 and FAS) in MFG were higher than in MG and MSC. In conclusion, the total RNA in MFG mainly derives from mammary epithelial cells and can be used to study the functional gene expression of mammary epithelial cells.

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The p100 coactivator is present in the nuclei of mammary epithelial cells and its abundance is increased in response to prolactin in culture and in mammary tissue during lactation

Journal of Endocrinology ◽

10.1677/joe.0.1710329 ◽

2001 ◽

Vol 171 (2) ◽

pp. 329-337 ◽

Cited By ~ 29

Author(s):

MK Broadhurst ◽

TT Wheeler

Keyword(s):

Epithelial Cells ◽

Cultured Cells ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Mammary Tissue ◽

Barr Virus ◽

Beta Casein ◽

Epstein Barr ◽

Lactation Cycle

The p100 coactivator, first identified as a coactivator of the Epstein-Barr virus-encoded transcription factor, EBNA-2, in cultured cells, interacts with a number of transcription factors. However, the role of p100 in animals is unclear. We found that the abundance of p100 is closely associated with the lactating state in mammary tissue of mice and cows. Using two antibodies against independent parts of the protein, p100 immunoreactivity was localised to mammary epithelial cells, and was enriched in both nuclei and endoplasmic reticulum/organelle fractions. Stimulation of beta-casein expression in cultured mammary epithelial cells was associated with an increase in abundance of the p100 protein. The relative abundance of p100 mRNA was not altered in mammary tissue throughout the gestation-lactation cycle, indicating that the abundance of p100 is altered by a post-transcriptional mechanism. Further work is required to clarify the function of p100 in mammary epithelial cells.

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Correction: Impact of Heat Stress on Cellular and Transcriptional Adaptation of Mammary Epithelial Cells in Riverine Buffalo (Bubalus Bubalis)

PLoS ONE ◽

10.1371/journal.pone.0191380 ◽

2018 ◽

Vol 13 (1) ◽

pp. e0191380 ◽

Cited By ~ 1

Author(s):

Neha Kapila ◽

Ankita Sharma ◽

Amit Kishore ◽

Monika Sodhi ◽

Pawan K. Tripathi ◽

...

Keyword(s):

Heat Stress ◽

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Bubalus Bubalis ◽

Mammary Epithelial ◽

Transcriptional Adaptation

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

ISRN Biotechnology ◽

10.5402/2013/735053 ◽

2013 ◽

Vol 2013 ◽

pp. 1-9 ◽

Cited By ~ 10

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.

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