Collection of Epithelial Cells from Rodent Mammary Gland Via Laser Capture Microdissection Yielding High-Quality RNA Suitable for Microarray Analysis

Rodent models of breast cancer continue to play a crucial role in discovering new approaches to treatment and prevention of the disease. Microarrays offer investigators the ability to screen thousands of genes per sample [1-5]. However, heterogeneous tissue may confound molecular analysis because it is currently impossible to discern which cells contribute which cellular constituents to a given tissue lysate. Laser capture microdissection (LCM) allows specific cell populations to be harvested, thus reducing the amount of biological noise while increasing the sensitivity of microarrays enabling detection of subtle differences in gene expression profiles among control and treatment groups [6]. Specific cell populations can be harvested from frozen or paraffin tissue sections using LCM [7-10]. However, RNA obtained from formalin-fixed paraffin-embedded tissue can be significantly degraded due to fragmentation and modification of the template through the addition of mono-methylol groups to the bases, thus interfering with RNA extraction and subsequent amplification [11-14]. Alternative methods of fixation and processing have been proposed for preservation of RNA in paraffin-embedded samples [15-17]. Nevertheless, they have not gained wide acceptance, and frozen tissue remains the gold standard for obtaining high-quality RNA.Obtaining suitable frozen sections for LCM from lipid-rich tissues presents many challenges, and rat mammary gland (MG) presents specific obstacles due to the high adipose content and relative low abundance of fibrous connective tissue as compared to human breast (Figure 1). Capturing the limited number of epithelial cells available in each MG sample is a race against time in order to preserve RNA integrity. While there are a growing number of articles on the subject of LCM, few have reported optimized conditions and detailed hands-on procedures to obtain high-quality RNA suitable for microarray analysis from rodent MG that yields a high percent call