Abstract
Assaying global cell cycle-regulated transcription in budding yeast involves extracting RNA from a synchronous population and proper normalization of detected transcript levels. Here, we describe synchronization of Saccharomyces cerevisiae cell populations by centrifugal elutriation, followed by the isolation of RNA for microarray analysis. Further, we outline the computational methods required to directly compare RNA abundance from individual time points within an experiment and to compare independent experiments. Together, these methods describe the complete workflow necessary to observe RNA abundance during the cell cycle.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Cho RJ, Campbell MJ, Winzeler EA, Steinmetz L, Conway A, Wodicka L, Wolfsberg TG, Gabrielian AE, Landsman D, Lockhart DJ, Davis RW (1998) A genome-wide transcriptional analysis of the mitotic cell cycle. Mol Cell 2(1):65–73
Orlando DA, Lin CY, Bernard A, Wang JY, Socolar JE, Iversen ES, Hartemink AJ, Haase SB (2008) Global control of cell-cycle transcription by coupled CDK and network oscillators. Nature 453(7197):944–947
Pramila T, Wu W, Miles S, Noble WS, Breeden LL (2006) The Forkhead transcription factor Hcm1 regulates chromosome segregation genes and fills the S-phase gap in the transcriptional circuitry of the cell cycle. Genes Dev 20(16):2266–2278
Spellman PT, Sherlock G, Zhang MQ, Iyer VR, Anders K, Eisen MB, Brown PO, Botstein D, Futcher B (1998) Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. Mol Biol Cell 9(12):3273–3297
Simmons Kovacs LA, Mayhew MB, Orlando DA, Jin Y, Li Q, Huang C, Reed SI, Mukherjee S, Haase SB (2012) Cyclin-dependent kinases are regulators and effectors of oscillations driven by a transcription factor network. Mol Cell 45(5):669–679
Walker GM (1999) Synchronization of yeast cell populations. Methods Cell Sci 21(2–3):87–93
Johnston LH, Johnson AL (1997) Elutriation of budding yeast. Methods Enzymol 283:342–350
Hartwell LH, Unger MW (1977) Unequal division in Saccharomyces cerevisiae and its implications for the control of cell division. J Cell Biol 75(2 Pt 1):422–435
Lord PG, Wheals AE (1980) Asymmetrical division of Saccharomyces cerevisiae. J Bacteriol 142(3):808–818
Lord PG, Wheals AE (1981) Variability in individual cell cycles of Saccharomyces cerevisiae. J Cell Sci 50:361–376
Woldringh CL, Huls PG, Vischer NO (1993) Volume growth of daughter and parent cells during the cell cycle of Saccharomyces cerevisiae a/alpha as determined by image cytometry. J Bacteriol 175(10):3174–3181
Orlando DA, Lin CY, Bernard A, Iversen ES, Hartemink AJ, Haase SB (2007) A probabilistic model for cell cycle distributions in synchrony experiments. Cell Cycle 6(4):478–488
Orlando DA, Iversen ES Jr, Hartemink AJ, Haase SB (2009) A branching process model for flow cytometry and budding index measurements in cell synchrony experiments. Ann Appl Stat 3(4):1521–1541
Chomczynski P, Sacchi N (2006) The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nat Protoc 1(2): 581–585
Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162(1):156–159
Perry RP, La Torre J, Kelley DE, Greenberg JR (1972) On the lability of poly(A) sequences during extraction of messenger RNA from polyribosomes. Biochim Biophys Acta 262(2):220–226
Collart MA, Oliviero S (2001) Preparation of yeast RNA. Curr Protoc Mol Biol Chapter 13:Unit13.12
Lim WK, Wang K, Lefebvre C, Califano A (2007) Comparative analysis of microarray normalization procedures: effects on reverse engineering gene networks. Bioinformatics 23(13):i282–i288
Gautier L, Cope L, Bolstad BM, Irizarry RA (2004) affy—analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 20(3):307–315
Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JY, Zhang J (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5(10):R80
R Core Team (2012) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0. http://www.R-project.org/
Li C, Wong WH (2001) Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection. Proc Natl Acad Sci U S A 98(1):31–36
Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP (2003) Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4(2):249–264
Hubbell E, Liu WM, Mei R (2002) Robust estimators for expression analysis. Bioinformatics 18(12):1585–1592
Acknowledgements
Members of the Haase Lab are thanked for their critical reading and comments.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Leman, A.R., Bristow, S.L., Haase, S.B. (2014). Analyzing Transcription Dynamics During the Budding Yeast Cell Cycle. In: Noguchi, E., Gadaleta, M. (eds) Cell Cycle Control. Methods in Molecular Biology, vol 1170. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0888-2_14
Download citation
DOI: https://doi.org/10.1007/978-1-4939-0888-2_14
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0887-5
Online ISBN: 978-1-4939-0888-2
eBook Packages: Springer Protocols