Cancer Prevention Laboratory, Colorado State University, 1173 Campus Delivery, Fort Collins, CO 80523, USA.
Biol Proced Online. 2010 Mar 3;12(1):9026. doi: 10.1007/s12575-010-9026-8.
Laser capture microdissection (LCM) enables collection of cell populations highly enriched for specific cell types that have the potential of yielding critical information about physiological and pathophysiological processes. One use of cells collected by LCM is for gene expression profiling. Samples intended for transcript analyses should be of the highest quality possible. RNA degradation is an ever-present concern in molecular biological assays, and LCM is no exception. This paper identifies issues related to preparation, collection, and processing in a lipid-rich tissue, rodent mammary gland, in which the epithelial to stromal cell ratio is low and the stromal component is primarily adipocytes, a situation that presents numerous technical challenges for high-quality RNA isolation. Our goal was to improve the procedure so that a greater probe set present call rate would be obtained when isolated RNA was evaluated using Affymetrix microarrays. The results showed that the quality of RNA isolated from epithelial cells of both mammary gland and mammary adenocarcinomas was high with a probe set present call rate of 65% and a high signal-to-noise ratio.
激光捕获显微切割（LCM）可用于收集高度富集特定细胞类型的细胞群体，这些细胞有可能提供有关生理和病理生理过程的关键信息。通过 LCM 收集的细胞的一种用途是进行基因表达谱分析。用于转录分析的样品应尽可能达到最高质量。RNA 降解是分子生物学检测中始终存在的问题，LCM 也不例外。本文确定了在富含脂质的组织（啮齿动物乳腺）中与准备、收集和处理相关的问题，其中上皮细胞与基质细胞的比例较低，基质成分主要是脂肪细胞，这种情况对高质量 RNA 分离提出了许多技术挑战。我们的目标是改进该程序，以便在用 Affymetrix 微阵列评估分离的 RNA 时获得更高的探针集存在率。结果表明，从乳腺和乳腺腺癌的上皮细胞中分离的 RNA 质量很高，探针集存在率为 65%，信噪比高。
