Synthesis of biotin-labeled RNA for gene expression measurements using oligonucleotide arrays

Ana E. Vázquez, Liping Nie, Ebenezer N. Yamoah

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
Pages21-29
Number of pages9
Volume493
DOIs
StatePublished - 2009

Publication series

NameMethods in Molecular Biology
Volume493
ISSN (Print)10643745

Keywords

  • Cochlea
  • Gene chips
  • Gene expression
  • Membranous labyrinth
  • Oligonucleotide microarrays

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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    Vázquez, A. E., Nie, L., & Yamoah, E. N. (2009). Synthesis of biotin-labeled RNA for gene expression measurements using oligonucleotide arrays. In Methods in Molecular Biology (Vol. 493, pp. 21-29). (Methods in Molecular Biology; Vol. 493). https://doi.org/10.1007/978-1-59745-523-7-2