Maintaining RNA integrity in a homogeneous population of mammary epithelial cells isolated by Laser Capture Microdissection

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.

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

“…Cryosectioning, staining, dehydration and harvesting of microdissected cells was performed as described Bevilacqua et al1. Total RNA was extracted from captured cells using the PicoPure® RNA Isolation Kit (Arcturus, Applied Biosystems) according to the manufacturer's instructions, including an on-column DNase I treatment (Qiagen).…”

“…They also form part of a complex and heterogeneous tissue along with multiple cell types including myoepithelial, stromal and immune cells, making it difficult to precisely assign gene expression to the different cell types. Thus, it is important to distinguish which cell populations play specific functions, and to obtain relevant expression profile data reflecting the in vivo state in order to recognize differences in these cell fractions and to obtain reliable transcriptomic results1. Mammary epithelial cells are normally subject to turnover and are replaced by new cells2.…”

“…Alternative sampling approaches include isolating RNA directly from biopsies of mammary gland tissue5, from somatic cells in milk that are naturally released during lactation67, from antibody-captured epithelial cells in milk8910 and from milk fat globules11. Also, laser-capture microdissection has been used to selectively isolate epithelial cells from frozen tissue sections of the mammary gland1. Some of these sampling procedures were validated in an earlier study using qPCR for specific genes and/or using microarray11.…”

Puberty is a critical window for the impact of diet on mammary gland development in the rabbit

“…In order to analyze the impact of diet during the peri‐pubertal period on mammary epithelial tissue, we assessed specific MEC gene expression patterns using laser capture microdissection (LCM). This approach was chosen in order to isolate and characterize the MEC population as the efficiency of this technique has already been proven in numerous tissues, species or during cancer studies . Stained mammary sections (Figure A) capturing MEC (Figure B) provided highly enriched material for mRNA studies.…”

Extraction of RNA from tough tissues with high proteoglycan content by cryosection, second phase separation and high salt precipitation

“…Cryosection was also tested since bovine NP tissue sections have been successfully prepared for histology. In laser capture microdissection (LCM), cryosectioning has been reported in preparing sections on glass or membrane slides [10][11][12][13][14][15]. With LCM, a specific region of interest can be selected for RNA extraction.…”