chapter  4
50 Pages

PCR-based methods for measuring transcript levels

As discussed in previous chapters, one of the recurring aims of molecular biology experiments is to visualize what is not ordinarily visible. In this book we are concerned with visualizing the products of gene expression: RNA and proteins. In visualizing them, we hope to be able to quantify them, if not absolutely, then at least relatively between amounts of a particular product in cell extracts from two experimental states. The example in the previous chapter was to label specific RNA or DNA sequences using hybridization so that they stand out from the background of other RNA molecules. The label is highly visible, so that even if the target RNA is low abundance, it will be seen. Furthermore, label incorporation is proportional to the amount of target RNA, so can be used to estimate its concentration. An alternative approach to the same problem of making a particular RNA molecule visible against a background of many other RNAs is to specifically increase the concentration of (amplify) the target RNA molecule to a level that becomes visible using gel electrophoresis and simple nucleic acid staining. If the concentration of amplification product were directly proportional to the amount of starting material, then the observed concentration of the product could be used to estimate the concentration of the original RNA species.