This chapter describes some of the methodological approaches that are used to study the involvement of nerve growth factor (NGF) in the neuroendocrine control of female sexual development. The ribonuclease protection assay is a highly sensitive and specific method for detection of tissue-specific mRNA expression. It utilizes a 32P-labeled antisense RNA probe that is first hybridized in solution to tissue mRNA and then subjected to digestion with ribonucleases. Hybridization of the probe to complementary mRNA sequences present in tissues makes the complex insensitive to ribonuclease digestion and results in a protected band that has the exact size of the hybridizing sequence. As complementary sense mRNA sequences can be synthesized by in vitro transcription, appropriate standard curves can be generated by dilution and used to quantitate the changes in tissue mRNA levels. Additionally, nerve growth factor protein is detection by two-site immunoassay. This method is basically that of Korsching and Thoenen, with less detail regarding the conceptual aspects of the assay, more detail in some technical aspects, and with specific focus on areas of the brain where NGF content is low.
|Original language||English (US)|
|Number of pages||18|
|Journal||Methods in Neurosciences|
|State||Published - Jan 1 1992|
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