Comparative quantitation of mRNA expression in the central nervous system using fluorescence in situ hybridization.
In situ hybridization to messenger RNA (mRNA) in complex tissues, such as the brain, allows the localization of gene expression to functionally distinct regions. It has been difficult to measure relative changes in gene expression within these regions because of the poor cellular resolution afforded by radioactively labeled probes and problems associated with densitometric analysis by counting silver grain deposition. Fluorescence in situ hybridization, using probes directly labeled with dyes that exhibit high quantum yield, provides both high-resolution localization of mRNA and high sensitivity for detection of hybridized probe. Digital image capture of fluorescence is readily amenable to densitometric analysis, thereby allowing relative quantification of mRNA expression in single cells or discrete brain nuclei. In this chapter, we describe protocols suitable for measuring relative changes in gene expression within individual cells of brain sections mounted on glass slides.
Day
Darren J
DJ
School of Biological Sciences, Victoria University, Wellington, New Zealand.
Mrkusich
Eli M
EM
Miller
John H
JH
D000818:Animals; D002490:Central Nervous System / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D015870:Gene Expression; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D015336:Molecular Probe Techniques; D011401:Promoter Regions, Genetic; D015347:RNA Probes / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014764:Viral Proteins / Q000235:genetics
D015347:RNA Probes; D012333:RNA, Messenger; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases