Conventional methods for RT-PCR involve obtaining samples by homogenizing whole or partial tissues in Trizol (Invitrogen), RNAzol (IsoTex Diagnostics), or similar reagents. While providing useful information about gene expression changes within organs of interest, results generated by these approaches are limited, as it is impossible to conclude which cell populations are responsible for the changes in gene expression. An additional shortcoming of this technique is that it results in the loss of microenvironments within the organ of interest. Therefore, inferences about which cells are making a factor, and which cells are responding to that factor are unable to be made.
Useful techniques to circumvent disruption of tissue structure in the analysis of gene expression are LCM and LDM. While they require specialized microscopes and systems, they are similar in that freshly-cut frozen tissue sections can be microdissected using either a general histological stain (like H&E) or by staining with fluorescently conjugated antibodies. The LCM system by Arcturus involves placing a plastic cap atop the tissue section of interest on a conventional microscope slide, and focusing and firing a laser at the bottom of the cap melting plastic directly onto the tissue region of interest. The cap is removed, and with it the tissue region of interest. One limitation of this technique is that it is greatly affected by humidity, which effects the melting of plastic onto the tissue. Leica’s LDM system requires placing frozen tissue sections on special slides featuring a plastic membrane on one side. A laser then melts the plastic membrane around the tissue region on interest, which frees it from the slide.
There is tremendous potential in these technologies, but there are a number of difficulties, especially when RT-PCR analysis is the end goal. (1) Any staining the tissue undergoes must be done quickly, to minimize RNA degradation. (2) Effective melting of plastic onto tissue or away from tissue (depending on LCM or LDM) requires dry, desiccated slides, which are conditions that are less than ideal for fluorescence. (3) These techniques have fairly small yields, and thus require a number of serial sections for detectable results by RT-PCR. In an effort to circumvent these concerns, staining methods must be fast, the dissection must done quickly, and on a high number of serial tissue sections.