Abstract
In the last decade, it became clear that transcription goes far beyond that of protein-coding genes. Most RNA molecules are transcribed from intergenic regions or introns and exhibit much variability in size, expression level, secondary structure, and evolutionary conservation. While for several types of non-coding RNAs some cellular functions have been reported, like for micro-RNAs and small nucleolar RNAs, for most others no indications of function or regulation have so far been found. Therefore, the RNA population inside a cell is diverse and cryptic and, thus, demands powerful methods to study its composition, abundance, and structure. DNA oligonucleotide microarrays have proven to be of great utility to study transcription of genes in various organisms. Recently, due to advancement in microarray technology, tiling microarrays that extend transcription measurement to genomic regions beyond protein-coding genes were designed for several species. The Saccharomyces cerevisiae yeast tiling array contains overlapping probes across the full genomic sequence, with consecutive probes starting every 8 bp on average on each strand, enabling strand-specific measurement of transcription from a full eukaryotic genome. Here, we describe the methods used to extract yeast RNA, convert it into first-strand cDNA, fragment, and label it for hybridization to the tiling array. This protocol will enable researchers not only to study which genes are expressed and to what levels, but also to identify non-coding RNAs and to study the structure of transcripts including their untranslated regions, alternative start, stop, and processing sites. This information will allow understanding their roles inside cells.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Amaral, P. P., Dinger, M. E., Mercer, T. R., and Mattick, J. S. (2008) The eukaryotic genome as an RNA machine. Science 319, 1787–1789.
Kapranov, P., Cawley, S. E., Drenkow, J., et al. (2002) Large-scale transcriptional activity in chromosomes 21 and 22. Science 296, 916–919.
Carninci, P., Kasukawa, T., Katayama, S., et al. (2005) The transcriptional landscape of the mammalian genome. Science 309, 1559–1563.
Birney, E., Stamatoyannopoulos, J. A., Dutta, A., et al. (2007) Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 447, 799–816.
Wodicka, L., Dong, H., Mittmann, M., Ho, M. H., and Lockhart, D. J. (1997) Genome-wide expression monitoring in Saccharomyces cerevisiae. Nat. Biotechnol. 15, 1359–1367.
Schena, M., Shalon, D., Davis, R. W., and Brown, P. O. (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467–470.
Lockhart, D. J., Dong, H., Byrne, M. C., et al. (1996) Expression monitoring by hybridization to high-density oligonucleotide arrays. Nat. Biotechnol. 14, 1675–1680.
Lashkari, D. A., DeRisi, J. L., McCusker, J. H., et al. (1997) Yeast microarrays for genome wide parallel genetic and gene expression analysis. Proc. Natl. Acad. Sci. USA 94, 13057–13062.
Fodor, S. P., Read, J. L., Pirrung, M. C., Stryer, L., Lu, A. T., and Solas, D. (1991) Light-directed, spatially addressable parallel chemical synthesis. Science 251, 767–773.
Yamada, K., Lim, J., Dale, J. M., et al. (2003) Empirical analysis of transcriptional activity in the Arabidopsis genome. Science 302, 842–846.
Tjaden, B., Saxena, R. M., Stolyar, S., Haynor, D. R., Kolker, E., and Rosenow, C. (2002) Transcriptome analysis of Escherichia coli using high-density oligonucleotide probe arrays. Nucleic Acids Res. 30, 3732–3738.
Selinger, D. W., Cheung, K. J., Mei, R., et al. (2000) RNA expression analysis using a 30 base pair resolution Escherichia coli genome array. Nat. Biotechnol. 18, 1262–1268.
David, L., Huber, W., Granovskaia, M., et al. (2006) A high-resolution map of transcription in the yeast genome. Proc. Natl. Acad. Sci. USA 103, 5320–5325.
Perocchi, F., Xu, Z., Clauder-Munster, S., and Steinmetz, L. M. (2007) Antisense artifacts in transcriptome microarray experiments are resolved by actinomycin D. Nucleic Acids Res. 35, e128.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Humana Press
About this protocol
Cite this protocol
David, L., Clauder-Münster, S., Steinmetz, L.M. (2011). Genome-Wide Transcriptome Analysis in Yeast Using High-Density Tiling Arrays. In: Castrillo, J., Oliver, S. (eds) Yeast Systems Biology. Methods in Molecular Biology, vol 759. Humana Press. https://doi.org/10.1007/978-1-61779-173-4_7
Download citation
DOI: https://doi.org/10.1007/978-1-61779-173-4_7
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-172-7
Online ISBN: 978-1-61779-173-4
eBook Packages: Springer Protocols