While there are numerous methods of quantitating levels of a specific RNA transcript utilizing blot hybridization technologies, this chapter focuses on the solution hybridization/nuclease protection assay because of its unique properties. These properties give distinct advantages for analyzing gene expression in the brain. Using conventional autoradiography techniques, one can readily quantitate levels as small as 50 fg of a specific RNA transcript, and with the use of phosphoimaging techniques, sensitivity can be taken down to at least another order of magnitude. Thus, this technique is quite useful for quantitating levels of transcripts that may be present at a very low copy number within the population of neurons under investigation, such as neurotransmitter receptors. This also helps identify the low abundance in nuclear transcripts involved in the biosynthesis of the specific mRNA. Secondly, because the size of the species being analyzed can be identified, numerous different RNAs of different sizes of protected fragments can be analyzed in a single sample. Ultimately, spliced RNAs can be identified based on which portions of the probe get protected or, on the other hand, multiple transcripts in the biosynthetic pathway can be identified.
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