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

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.

Figure 1. Total RNA capillary electrophoresis chromatograms from mammary and milk fractions.

(A) Mammary tissue (MGT), (B) Somatic cells (SC), (C) Laser capture mammary epithelial cells (LCMEC), (D) Antibody-captured milk mammary epithelial cells (mMEC), (E) Milk fat globule (MFG). Traces (A) and (C) (Agilent Bioanalyzer), traces (B), (D), (E) Experion Analyzer (BioRad). These diagrams show the differences in quality of RNA obtained from the different fractions.

Figure 2

Mammary gland tissue (MGT) before microdissection (A) and mammary epithelial cells after laser microdissection (LCMEC) (B1 and B2). Chromatograms from total RNA capillary electrophoresis; MGT before microdissection (A1) LCMEC after microdissection (B3).