Isolating individual cells for molecular analysis
High-throughput technology is continually being invented and refined to allow increasingly sensitive molecular analysis of tissue samples. As new cutting-edge microanalytical procedures and instruments are developed, more rigorous demands will be placed on sample preparation techniques. Using laser-capture microdissection (LCM) in combination with these sensitive analytical methods, researchers can obtain more accurate data and address previously unanswerable questions.
The power of proteomic and genomic techniques relies heavily on the preparation of homogeneous cell populations. Although flow cytometry has long been used to enrich a particular cell type in suspension, the ability to purify cells from solid tissue samples, such as biopsies, has lagged. Another disadvantage of flow cytometry is the requirement of a specific marker for selection. To improve cell isolation, several microdissection methods have been developed, but these are often time-consuming and imprecise.
As an alternative, Michael Emmert-Buck and colleagues at the National Institutes of Health (NIH) in Bethesda, MD, developed LCM and demonstrated its usefulness for studying various tissue types at the DNA, mRNA, and protein levels. The NIH group eventually joined forces with Arcturus Engineering, Inc. (Mountain View, CA; www.arctur.com), as part of a Collaborative Research and Development Agreement for the commercialization of this technology. In addition to its elegant simplicity, LCM has the advantage of preserving the tissues original morphology while avoiding contamination from surrounding tissue. Samuel Chu, technical support specialist at Arcturus, explains, The benefits of microdissection include the ability to procure a pure population of cells that would give y
What is LCM?
After the appropriate cells have been selected, the film and adherent cells are removed, and the unselected tissue remains in contact with the glass slide. (For QuickTime movies of this process, visit www.arctur.com/lcm_movies.html.) These cells can then be subjected to appropriate extraction conditions for ensuing molecular analysis. To improve the convenience of the technique, the transfer film can be mounted on a transparent cap that fits on a 500-µL microcentrifuge tube.
LCM is compatible with various common methods for the preparation of tissue sections. Tissues are typically fixed by alcohol-based precipitation techniques. Aldehyde-based fixation may also be used, but covalent cross-linking of macromolecules can potentially interfere with subsequent analysis of RNA or protein. Sections of 6-mm thickness can then be prepared from paraffin-embedded or frozen tissue and mounted on glass slides. These sections may be stained by standard techniques such as hematoxylin and eosin, methylene green nuclear stain, fluorescence in situ hybridization, or immunohistochemistry for identification of tissue morphology and cell populations of interest. Because the section thickness is less than that of a cell, up to 20 cells may need to be selected to obtain a complete genome or expression profile. Up to 3000 transfers can be performed on one film, representing more than 6000 cells, depending on their size. The cells are then lysed and extracted in an appropriate buffer for the analysis of DNA, RNA, or protein (see Figure 2). Remarkably, single cells captured by this technique have been successfully analyzed by techniques based on nucleic acid amplification.
The major disadvantage of LCM is that it isolates minute amounts of material, which limits analysis to amplification-based techniques or collection of numerous cells, as in protein analysis. Although this technique is faster than previous microdissection methods, isolation of large numbers of cells from many sections can require considerable time. Another disadvantage is that cover slips and mounting solutions are not compatible with LCM a
Complementary to these DNA and RNA analyses is the examination of differential protein-expression profiles by SDS-PAGE and 2D-PAGE. Mass spectrometric sequencing, peptide mass fingerprinting, in-gel zymography, or Western blot has then been used to identify proteins of interest. Newly developed protein chips may also prove useful to this end. Protein analysis of microdissected tissue can provide important information not accessible with nucleic acid-based techniques, including protein stability and translation efficiency, post-translational modification, proteinprotein interaction, and proteinDNA interaction.
Combining LCM with several of the genomic and proteomic techniques mentioned above, the Cancer Genome Anatomy Project (www.ncbi.nlm.nih.gov/CGAP) at the NIH has established an effort to document the progression of normal cells to premalignant and metastatic cancer cells in various tissues. This NIH-based initiative is taking a five-sided approach to this problem. These approaches will use cDNA microarrays to identify genes expressed during mouse and human tumor development, 2D-PAGE and mass spectrometry to profile molecular differences between normal and diseased tissue, and PCR-based techniques to identify genetic polymorphisms and chromosomal aberrations.
Besides their application to disease research, microdissected sections from genetic model organisms such as flies, worms, zebrafish, and mice may lead to new insights in developmental biology. Comparison of gene expression patterns in neighboring cells can be analyzed to improve our understanding of cell fate and cell differentiation. Similarly, differential expression between cell types in particular tissues has already been examined, as in the case of adrenomedullary and adrenocortical cells. LCM is still in its infancy, and new applications are continuing to be explored, Chu states.