Analyzing transcription dynamics during the budding yeast cell cycle.

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.

[1]  Wei-Min Liu,et al.  Robust estimators for expression analysis , 2002, Bioinform..

[2]  William Stafford Noble,et al.  The Forkhead transcription factor Hcm1 regulates chromosome segregation genes and fills the S-phase gap in the transcriptional circuitry of the cell cycle. , 2006, Genes & development.

[3]  Allister Bernard,et al.  A Probabilistic Model for Cell Cycle Distributions in Synchrony Experiments , 2007, Cell cycle.

[4]  Benjamin M. Bolstad,et al.  affy - analysis of Affymetrix GeneChip data at the probe level , 2004, Bioinform..

[5]  Ronald W. Davis,et al.  A genome-wide transcriptional analysis of the mitotic cell cycle. , 1998, Molecular cell.

[6]  C. Woldringh,et al.  Volume growth of daughter and parent cells during the cell cycle of Saccharomyces cerevisiae a/alpha as determined by image cytometry , 1993, Journal of bacteriology.

[7]  Joshua E. S. Socolar,et al.  Global control of cell-cycle transcription by coupled CDK and network oscillators , 2008, Nature.

[8]  G. Walker.  Synchronization of yeast cell populations. , 1999, Methods in cell science : an official journal of the Society for In Vitro Biology.

[9]  L. Hartwell,et al.  Unequal division in Saccharomyces cerevisiae and its implications for the control of cell division , 1977, The Journal of cell biology.

[10]  Michael Ruogu Zhang,et al.  Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. , 1998, Molecular biology of the cell.

[11]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.

[12]  Michael B. Mayhew,et al.  Cyclin-dependent kinases are regulators and effectors of oscillations driven by a transcription factor network. , 2012, Molecular cell.

[13]  P. Lord,et al.  Variability in individual cell cycles of Saccharomyces cerevisiae. , 1981, Journal of cell science.

[14]  David A. Orlando,et al.  A branching process model for flow cytometry and budding index measurements in cell synchrony experiments. , 2010, The annals of applied statistics.

[15]  M. Collart,et al.  Preparation of Yeast RNA , 1993, Current protocols in molecular biology.

[16]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[17]  C. Li,et al.  Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[18]  Elutriation of budding yeast. , 1997, Methods in enzymology.

[19]  Jean YH Yang,et al.  Bioconductor: open software development for computational biology and bioinformatics , 2004, Genome Biology.

[20]  L. Hartwell,et al.  Asymmetrical division of Saccharomyces cerevisiae , 1980, Journal of bacteriology.

[21]  P. Chomczyński,et al.  The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on , 2006, Nature Protocols.

[22]  J R Greenberg,et al.  On the lability of poly(A) sequences during extraction of messenger RNA from polyribosomes. , 1972, Biochimica et biophysica acta.

[23]  Kai Wang,et al.  Comparative analysis of microarray normalization procedures: effects on reverse engineering gene networks , 2007, ISMB/ECCB.

[24]  Rafael A Irizarry,et al.  Exploration, normalization, and summaries of high density oligonucleotide array probe level data. , 2003, Biostatistics.