ABSTRACT
Molecular biology has greatly impacted the field of neuroendocrinology by facilitating the identification of receptor subtypes that otherwise might be difficult to detect using traditional receptor binding methodologies. This chapter describes some of the considerations surrounding the design and optimization of a successful semi-quantitative or quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay, and summarizes some important concerns for reliable measurement of neuroendocrine parameters. RT-PCR reactions start with RNA and require the sequential use of the reverse transcriptase and PCR reactions. In many RT-PCR reactions, multiple primer pairs may be added to amplify different genes such as the target gene and the control gene. Using RT-PCR in quantitative measurements necessitates the use of an internal control. The controls are co-amplified in the same samples as target gene and monitor variations in the RT-PCR reactions as well as serve as standards for quantitation. There are two types of controls that are used for the quantitation of RT-PCR products: endogenous and exogenous standards.
