Comparison of five different RNA sources to examine the lactating bovine mammary gland transcriptome using RNA-Sequencing

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Abstract

The objective of this study was to examine five different sources of RNA, namely mammary gland tissue (MGT), milk somatic cells (SC), laser microdissected mammary epithelial cells (LCMEC), milk fat globules (MFG) and antibody-captured milk mammary epithelial cells (mMEC) to analyze the bovine mammary gland transcriptome using RNA-Sequencing. Our results provide a comparison between different sampling methods (invasive and non-invasive) to define the transcriptome of mammary gland tissue and milk cells. This information will be of value to investigators in choosing the most appropriate sampling method for different research applications to study specific physiological states during lactation. One of the simplest procedures to study the transcriptome associated with milk appears to be the isolation of total RNA directly from SC or MFG released into milk during lactation. Our results indicate that the SC and MFG transcriptome are representative of MGT and LCMEC and can be used as effective and alternative samples to study mammary gland expression without the need to perform a tissue biopsy.

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Transcriptional profiling of bovine milk using RNA sequencing

Saumya Wickramasinghe, Gonzalo Rincón, Alma Islas–Trejo … (2012)

Background Cow milk is a complex bioactive fluid consumed by humans beyond infancy. Even though the chemical and physical properties of cow milk are well characterized, very limited research has been done on characterizing the milk transcriptome. This study performs a comprehensive expression profiling of genes expressed in milk somatic cells of transition (day 15), peak (day 90) and late (day 250) lactation Holstein cows by RNA sequencing. Milk samples were collected from Holstein cows at 15, 90 and 250 days of lactation, and RNA was extracted from the pelleted milk cells. Gene expression analysis was conducted by Illumina RNA sequencing. Sequence reads were assembled and analyzed in CLC Genomics Workbench. Gene Ontology (GO) and pathway analysis were performed using the Blast2GO program and GeneGo application of MetaCore program. Results A total of 16,892 genes were expressed in transition lactation, 19,094 genes were expressed in peak lactation and 18,070 genes were expressed in late lactation. Regardless of the lactation stage approximately 9,000 genes showed ubiquitous expression. Genes encoding caseins, whey proteins and enzymes in lactose synthesis pathway showed higher expression in early lactation. The majority of genes in the fat metabolism pathway had high expression in transition and peak lactation milk. Most of the genes encoding for endogenous proteases and enzymes in ubiquitin-proteasome pathway showed higher expression along the course of lactation. Conclusions This is the first study to describe the comprehensive bovine milk transcriptome in Holstein cows. The results revealed that 69% of NCBI Btau 4.0 annotated genes are expressed in bovine milk somatic cells. Most of the genes were ubiquitously expressed in all three stages of lactation. However, a fraction of the milk transcriptome has genes devoted to specific functions unique to the lactation stage. This indicates the ability of milk somatic cells to adapt to different molecular functions according to the biological need of the animal. This study provides a valuable insight into the biology of lactation in the cow, as well as many avenues for future research on the bovine lactome.

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Adipophilin is a specific marker of lipid accumulation in diverse cell types and diseases.

Hans W. Heid, Brendan T Keenan, Ashley Moll … (1998)

We report the human DNA and protein sequence of adipophilin and its association with the surface of lipid droplets. The amino acid sequence of human adipophilin has been determined by using cDNA clones from several tissues and confirmed by the reverse transcription/polymerase chain reaction method and Edman sequencing. The open reading frame of adipophilin encodes a polypeptide with a calculated molecular weight of 48.1 kDa and an isoelectric point of 6.72. By immunofluorescence and electron-microscopic localization with newly raised specific poly- and monoclonal antibodies, we show that this protein is not restricted to adipocytes as previously indicated by studies of the mouse homologous protein, adipose-differentiation-related protein. Adipophilin occurs in a wide range of cultured cell lines, including fibroblasts and endothelial and epithelial cells. In tissues, however, expression of adipophilin is restricted to certain cell types, such as lactating mammary epithelial cells, adrenal cortex cells, Sertoli and Leydig cells of the male reproductive system, and steatosis or fatty change hepatocytes in alcoholic liver cirrhosis. Our results reveal adipophilin as a possible new marker for the identification of specialized differentiated cells containing lipid droplets and for diseases associated with fat-accumulating cells.

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An improved method for the routine biopsy of bovine mammary tissue.

Tessa Davis, K Stelwagen, Thomas McFadden … (1996)

Eight primiparous cows in midlactation were used to determine a method for the mammary biopsy of standing cows in full lactation. Cows were mildly sedated; therefore, preoperative feed withdrawal was not necessary. A core of secretory tissue (0.75 to 1 g) was extracted using a rotating stainless steel cannula with a retractable blade at the cutting edge. Postoperative recovery was rapid, taking only 15 min per cow, and the method was reliable and efficient. The presence of secretory tissue was verified by histology and in situ hybridization with alpha s1-casein and alpha-lactalbumin probes. The capsular end of the core contained more connective tissue, and the parenchyma showed heterogeneous expression of alpha s1-casein and alpha-lactalbumin. Despite some postoperative bleeding, milk yield and composition in the biopsied gland were affected only transiently. Yield recovered by 3.5 d after biopsy, and composition recovered by 6.5 d after biopsy. Yield and composition of milk from the control glands were not affected by the procedure. Biopsy sites healed rapidly and without infection. No clinical mastitis was observed in any of the biopsied cows throughout the remainder of the lactation.

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Journal

Journal ID (nlm-ta): Sci Rep

Journal ID (iso-abbrev): Sci Rep

Title: Scientific Reports

Publisher: Nature Publishing Group

ISSN (Electronic): 2045-2322

Publication date (Electronic): 08 July 2014

Publication date Collection: 2014

Volume: 4

Electronic Location Identifier: 5297

Affiliations

[1 ]Department of Animal Science, University of California-Davis , One Shields Ave., Davis, 95616, CA, USA

[2 ]Institut National de la Recherche Agronomique , UMR 1313 Génétique animale et Biologie intégrative, F-78350 Jouy-en-Josas, France

[3 ]INRA, AGROCAMPUS OUEST , UMR1348 PEGASE, F-35590 Saint-Gilles, France

[4 ]These authors contributed equally to this work.

Author notes

[a ] patrice.martin@ 123456jouy.inra.fr

[b ] jfmedrano@ 123456ucdavis.edu

Article

Publisher Item ID: srep05297

DOI: 10.1038/srep05297

PMC ID: 5381611

PubMed ID: 25001089

SO-VID: b7789846-9e2f-406a-a11d-4bf81bab1b52

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This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder in order to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/

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Date received : 10 October 2013

Date accepted : 23 April 2014

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Comparison of five different RNA sources to examine the lactating bovine mammary gland transcriptome using RNA-Sequencing

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Most cited references 18

Transcriptional profiling of bovine milk using RNA sequencing

Adipophilin is a specific marker of lipid accumulation in diverse cell types and diseases.

An improved method for the routine biopsy of bovine mammary tissue.

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