Gene Expression Analysis

The analysis of tissue- or cell-specific gene expression is of paramount importance for many fields of biological and biomedical research. However, tissue heterogeneity can make it very difficult to assign gene expression profiles or specific messages to defined cell populations if gross tissue extracts serve as a source for mRNA, and significant efforts have been put into developing microdissection strategies which allow cell- or tissue-specific gene expression analysis. In contrast to DNA, mRNA

is more sensitive to fixation, is quickly degraded by ubiquitous RNases, and requires stringent RNase-free conditions during specimen handling. Keeping these requirements in mind, mRNA of good quality can be recovered from microdissected frozen tissue samples down to the single-cell level, suitable for RT-PCR or quantitative real-time RT-PCR.[5] Rapid, optimized im-munohistochemical or immunofluorescence staining protocols allow the combination of phenotypical identification of target cells with mRNA analysis.[6,7] The superior speed of LCM and the fact that capturing is performed from dried slides allow sampling of large numbers of cells without significant RNA degradation during the procurement procedure. If very small numbers of cells serve as template or if large-scale expression profiling using cDNA or oligonucleotide arrays is used, random mRNA amplification protocols can be em-ployed.[8] In most studies, T7 RNA polymerase-based strategies have been the preferred technique in order to achieve an unbiased mRNA amplification from small amounts of tissue. Array-based expression screening of LCM-captured tissues has been used successfully for delineating expression differences of microdissected small and large neurons from dorsal root ganglia[9] or expression changes during breast cancer progression,1-10-1 to name only a few examples.

Another application of LCM is the generation of expression libraries from purified cell populations, which has been performed at a large scale for the cancer genome anatomy project (CGAP; http://www.ncbi.nlm.nih.gov/ ncicgap) sponsored by the National Cancer Institute.[11] This approach is intended to give a more faithful representation of gene expression patterns in epithelial neoplasms at all stages of development, in contrast to established cell lines which are usually derived from terminal-stage cancer patients, and in addition may show the artefacts of prolonged culture and the lack of an accompanying tissue microenvironment.

Although frozen tissue is the preferred source for mRNA analysis, the vast amounts of available archival specimens make paraffin-embedded tissues a valuable resource, and several groups have demonstrated that RNA derived from formalin-fixed paraffin blocks is suitable for certain types of analysis, such as conventional and real-time RT-PCR. A few hundred cells from paraffin sections render enough RNA for several RT-PCR assays without the need for preamplification steps, provided that the amplified fragment is below 100-120 bp.[12] Using the TaqMan technology with fluorescence-labeled probes, highly reproducible expression values can be obtained from microdissected archival material over a broad range of starting material and template concentrations.[13] The precision achievable by combining target cell purification by LCM with quantitative

RT-PCR and the ease with which these two techniques can be applied to paraffin sections make this approach a valuable tool for advanced molecular diagnostics.

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