Abstract
BackgroundLaser-capture microdissection (LCM) that enables the isolation of specific cell populations from complex tissues under morphological control is increasingly used for subsequent gene expression studies in cell biology by methods such as real-time quantitative PCR (qPCR), microarrays and most recently by RNA-sequencing. Challenges are i) to select precisely and efficiently cells of interest and ii) to maintain RNA integrity. The mammary gland which is a complex and heterogeneous tissue, consists of multiple cell types, changing in relative proportion during its development and thus hampering gene expression profiling comparison on whole tissue between physiological stages. During lactation, mammary epithelial cells (MEC) are predominant. However several other cell types, including myoepithelial (MMC) and immune cells are present, making it difficult to precisely determine the specificity of gene expression to the cell type of origin. In this work, an optimized reliable procedure for producing RNA from alveolar epithelial cells isolated from frozen histological sections of lactating goat, sheep and cow mammary glands using an infrared-laser based Arcturus Veritas LCM (Applied Biosystems®) system has been developed. The following steps of the microdissection workflow: cryosectioning, staining, dehydration and harvesting of microdissected cells have been carefully considered and designed to ensure cell capture efficiency without compromising RNA integrity.ResultsThe best results were obtained when staining 8 μm-thick sections with Cresyl violet® (Ambion, Applied Biosystems®) and capturing microdissected cells during less than 2 hours before RNA extraction. In addition, particular attention was paid to animal preparation before biopsies or slaughtering (milking) and freezing of tissue blocks which were embedded in a cryoprotective compound before being immersed in isopentane. The amount of RNA thus obtained from ca.150 to 250 acini (300,000 to 600,000 μm2) ranges between 5 to 10 ng. RNA integrity number (RIN) was ca. 8.0 and selectivity of this LCM protocol was demonstrated through qPCR analyses for several alveolar cell specific genes, including LALBA (α-lactalbumin) and CSN1S2 (αs2-casein), as well as Krt14 (cytokeratin 14), CD3e and CD68 which are specific markers of MMC, lymphocytes and macrophages, respectively.ConclusionsRNAs isolated from MEC in this manner were of very good quality for subsequent linear amplification, thus making it possible to establish a referential gene expression profile of the healthy MEC, a useful platform for tumor biomarker discovery.
Highlights
Laser-capture microdissection (LCM) that enables the isolation of specific cell populations from complex tissues under morphological control is increasingly used for subsequent gene expression studies in cell biology by methods such as real-time quantitative PCR, microarrays and most recently by RNA-sequencing
Milk is synthesized in mammary gland during lactation and though this process has been thoroughly studied, we still do not know precisely what mechanisms are involved in the intracellular transport and secretion of milk components, including supra-molecular structures, such as casein micelles [1,2] which are assembled during their transit within the mammary epithelial cell (MEC)
Given that recent studies [36,37,38] reported the effects of tissue manipulation on RNA quality and gene expression, and that each tissue requires specific protocol for reliable results, we have evaluated the impact of the main critical steps during slide preparation and capture of MEC
Summary
Laser-capture microdissection (LCM) that enables the isolation of specific cell populations from complex tissues under morphological control is increasingly used for subsequent gene expression studies in cell biology by methods such as real-time quantitative PCR (qPCR), microarrays and most recently by RNA-sequencing. The mammary gland which is a complex and heterogeneous tissue, consists of multiple cell types, changing in relative proportion during its development and hampering gene expression profiling comparison on whole tissue between physiological stages. Epithelial cells are predominant relative to adipocytes which are more abundant in the nulliparous gland [3]. Since both cell types are involved in lipid metabolism using the same metabolic pathways and enzymes, it becomes difficult to sort out the function of each cell type [4,5]
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