ABSTRACT

Some of the putative sleep-promoting compounds, e.g., interleukin-1β, are produced by action of a single gene, while the levels of other compounds, such as adenosine, are determined by a complex set of interrelated enzymes, all of which can be transcriptionally regulated. Thus, no single paradigm is likely to prove applicable to all putative sleep-promoting compounds. The estimation of messenger ribonucleic acid (mRNA) level and its changes have been accomplished by the DNA/RNA or RNA/RNA hybridization methods. Northern blot and nuclease protection assays have been used most extensively. Reverse transcription (RT)–polymerase chain reaction (PCR) is a very sensitive and straightforward method of mRNA analysis. RT–PCR is a two-step process. Both steps must be considered in quantitation of mRNA. In practice, however, the second step poses the more serious barrier in acquiring quantitative data. Because of the exponential character of the PCR, small changes in amplification efficiency produce considerable differences in the reaction yield.