chapter  2
60 Pages

Isolation and analysis of RNA

If you want to study the control of gene expression, at some stage you will have to measure transcript levels. Much can happen to a transcript before it is translated into an easily assayable reporter enzyme (Chapter 6), and so to get a true picture of the dynamics of transcription, RNA levels are the place to look. The problem is, RNA molecules are not designed to hang around long enough for scientists to sit and count them. They facilitate rapid responses to external stimuli; they are made and degraded rapidly, and in many cases this property confounds our attempts to catch a fleeting glimpse. The only way to work with transcripts is to put them into a sort of suspended animation. Using various chemicals, one can purify and stabilize RNA molecules long enough to quantify them. However, it should be said from the start that this is an artificial situation, and the fact that different RNA molecules respond to stabilization in different ways is the primary reason why measuring transcript levels can, in some experiments, be a matter of general trends rather than absolute numerical accuracy.