Gene Quantification: Choosing the Target

Abstract

Before going to great effort (and expense) trying to localize a gene of interest in a particular cell or tissue, one must determine adequate positive and negative control cells or tissues. As already mentioned, each cell and each tissue may require different conditions using the techniques described in this book and elsewhere [1]. Screening samples for the target of interest is essential prior to performing critical experiments on study samples. The methods of screening vary according to the target and dependent upon the detection of either RNA or DNA. Samples fall into categories we call the 4Fs: fresh, fixed, frozen, or f.....(forgotten). Frozen samples can either be frozen dry, frozen in freezing media (cells), or frozen in freezing compound (tissue). In any case, it would be useful to have the techniques to screen all of these tissues prior to in situ analysis. The goal of this chapter is to describe and illustrate useful methods to screen fresh tissue, frozen tissue, or tissue embedded in blocks for subsequent in situ analysis. Figure 4.1 illustrates how specific parts of the tissue sample can be screened using the methods to be described. The tissue in paraffin or freezing compound can be scored with a scalpel blade prior to sectioning to produce scrolls from specific areas of the tissue. Negative control tissue from adjacent normal areas minimizes the effects of block to block variation when choosing control tissues.