Synthesis of biotin-labeled RNA for gene expression measurements using oligonucleotide arrays.
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 approximately 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 immunohistochemical 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.
Department of Otolaryngology, Center for Neuroscience University of California, Davis, CA, USA.
Ana E
AE
Vázquez
Liping
L
Nie
Ebenezer N
EN
Yamoah
D001710:Biotin / Q000737:chemistry*; D007758:Ear, Inner / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012313:RNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D012313:RNA; D001710:Biotin