Maintaining RNA integrity in a homogeneous population of mammary epithelial cells isolated by Laser Capture Microdissection.
BMC Cell Biol. 2010-12-01; 11(1):
DOI: 10.1186/1471-2121-11-95
Read on PubMed
BACKGROUND: 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. 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.
RESULTS: The 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.
CONCLUSIONS: RNAs 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.