Laser Capture Microdissection of Single Cells from Complex Tissues

Abstract

Laser capture microdissection (LCM) is a new method used to select and procure cell clusters from tissue sections. Once captured, the DNA, RNA or protein can be easily extracted from the isolated cells and analyzed by conventional PCR, reverse transcription (RT)-PCR or polyacrylamide gel electrophoresis, including protein zymography for specific macromolecular changes. In LCM, a thermoplastic polymer coating [ethylene vinyl acetate (EVA)] attached to a rigid support is placed in contact with a tissue section. The EVA polymer over microscopically selected cell clusters is precisely activated by a near-infrared laser pulse and then bonds to the targeted area. Removal of the EVA and its support from the tissue section procures the selected cell aggregates for molecular analysis. This initial NIH LCM approach using a flat transfer EVA film has been recently commercialized and has proven to be an effective routine microdissection technique for subsequent macromolecular analysis in many laboratories around the world. However, reliable and precise capture of individual cells from tissue sections has been difficult to perform with the current LCM instruments. In this report, we describe the capture of individual cells with a new NIH LCM microscope, which epi-irradiates the EVA polymer overlying individual cells with 1-ms laser pulses focused to 6 microns. A computer-controlled arm precisely positions a 40-micron-wide strip of a cylindrical EVA surface onto a sample with a light contact force (ca. 0.1 g). The small contact force and contact area on the film on the sample diminishes nonspecific transfer to negligible levels. By slightly rotating the cylinder to provide a renewable transfer surface, concentration of a distinct cell type on a single cylinder is possible. Using this novel adaptation, we demonstrate the rapid and practical capture of single cells from different types of tissue sections, including immunostained cells.

Single-Cell Gene Expression Analysis: Implications for Neurodegenerative and Neuropsychiatric Disorders

“…Individual cells and pooled populations of similar cell types are visualized by immunocytochemical and/or histochemical procedures for optimal identification of specific cells of interest (3,34). RNA, DNA, and protein extraction methods can be performed on microdissected cells (37)(38)(39)(40), although LCM is primarily used for RNA extraction and subsequent cDNA microarray analysis. Control experiments for single-cell analysis include no input of RNA (no template control) and RNase pretreatment of tissue sections prior to LCM of specific cells (2,3,34).…”

Lymphocytic vasculitis in X-linked lymphoproliferative disease

“…Therefore, we chose to search for viral DNA by PCR. Although the vascular infiltrate was composed primarily of T cells and the latent EBV genome resides mainly within B lymphocytes, 14 we used LCM 15 to separate vessel wall from infiltrate to discount the possibility of significant B-cell contamination ( Figure 4A-C). We were able to detect the presence of the EBV genome in both the vascular tissue and the testes by PCR (Figure 4D), suggesting a widespread distribution of EBV, including a presence in vascular tissue.…”

Laser capture microdissection and single-cell RT-PCR without RNA purification

“…Extension of LCM from small populations of cells to individual cells has relied on PCR amplification of cell DNA (Suarez-Quian et al, 1999;Obiakor et al, 2002;Orba et al, 2003) or the perceived need to purify the very small amount of RNA, approximately 20 pg from a single captured cell for RT and PCR (Jin et al, 2001;Parlato et al, 2002;Michel et al, 2003;Kamme et al, 2004;Lu et al, 2004;Fassunke et al, 2004). Hydraulic microdissection has also been coupled with nested-PCR amplification of genomic DNA to identify sequences in single B cells (Obiakor et al, 2002).…”