2. Synthesis of Biotin-Labeled RNA for Gene Expression Measurements Using Oligonucleotide Arrays
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Using gene arrays, it is currently possible to simultaneously measure mRNA levels of many genes in any tissue of interest.
Undoubtedly, comprehensive measurements of gene expression as part of carefully designed experiments will continue to further
our understanding of audition and have the potential to open up new avenues of research. This chapter describes a reliable
protocol to prepare high-quality biotin-labeled RNA target, specifically for oligonucleotide array experiments. The procedure
includes isolation of high-quality total RNA, synthesis of double-stranded cDNA engineered for in vitro transcription with
T7 RNA polymerase, subsequent in vitro transcription in the presence of biotin-labeled ribonucleotides, and fractionation
of the RNA to ∼ 500 bp fragments, suitable for oligonucleotide array experiments. Because the membranous labyrinth is composed
of functionally interdependent cellular structures, which themselves contain numerous, highly differentiated cell types, comprehensive
analysis of gene expression in the cochlea is best complemented by immunohistotochemical studies or, if no suitable antibodies
are available, by in situ hybridization studies. Either one of these techniques will identify the specific cell types that
express the genes of interests.
Affiliation(s): (3) Department of Otolaryngology, Center for Neuroscience University of California, Davis, CA, USA
Series: Methods in Molecular Biology | Volume: 493 | Pub. Date: Oct-28-2008 | Page Range: 21-29 | DOI: 10.1007/978-1-59745-523-7_2
Subject: Molecular Medicine
Key Words: Gene expression - oligonucleotide microarrays - gene chips - membranous labyrinth - cochlea
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