ABSTRACT
Sleep presents challenges that should be carefully considered both prior to implementing molecular biological techniques and in the conclusions that may be drawn from the results. Specific messenger ribonucleic acids (mRNA) that are expressed either in sleep or wakefulness have been found. However, these mRNAs were found in the cortex. If mRNA expression can be differentially monitored using in situ hybridization, it could be the technique of choice for use in sleep studies. The chapter utilises northern blot analysis, in situ hybridization, and semiquantitative reverse transcription–polymerase chain reaction to monitor steady-state levels of specific mRNAs. Northern hybridization is a technique used to determine the amount, size, and integrity of messenger mRNA, the RNA which encodes protein. The in situ hybridization provides cellular localization of the message. Using an internal standard allows one to screen a pool of marker mRNAs from a single total RNA sample in a relatively straightforward manner.
