title: Comparison of sampling techniques for parallel analysis of transcript and metabolite levels in Saccharomyces cerevisiae.
abstract: Mathematical modelling of cellular processes is crucial for the understanding of the cell or organism as a whole. Genome-wide observations, at the levels of the transcriptome, proteome and metabolome, provide a high coverage of the molecular constituents of the system in study. Time-course experiments are important for gaining insight into a system's dynamics and are needed for mathematical modelling. In time-course experiments it is crucial to use efficient and fast sampling techniques. We evaluated several techniques to sample and process yeast cultures for parallel analysis of the transcriptome and metabolome. The evaluation was made by measuring the quality of the RNA obtained with UV-spectroscopy, capillary electrophoresis and microarray hybridization. The protocol developed involves rapid collection by spraying the sample into -40 degrees C tricine-buffered methanol (as previously described for yeast metabolome analysis), followed by the separation of cells from the culture medium in low-temperature rapid centrifugation. Removal of the residual methanol is carried out by freeze-drying the pellet at -35 degrees C. RNA and metabolites can then be extracted from the same freeze-dried sample obtained with this procedure.
authors.affiliation: Virginia Bioinformatics Institute, Washington Street, Blacksburg, VA 24061-0477, USA.
authors.forename: Ana Margarida
authors.initials: AM
authors.lastname: Martins
authors.forename: Wei
authors.initials: W
authors.lastname: Sha
authors.forename: Clive
authors.initials: C
authors.lastname: Evans
authors.forename: Susan
authors.initials: S
authors.lastname: Martino-Catt
authors.forename: Pedro
authors.initials: P
authors.lastname: Mendes
authors.forename: Vladimir
authors.initials: V
authors.lastname: Shulaev
mesh_terms: D002498:Centrifugation; D003451:Cryoprotective Agents; D005615:Freezing; D005998:Glycine / Q000031:analogs & derivatives; D000432:Methanol; D012331:RNA, Fungal / Q000032:analysis / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000302:isolation & purification*
chemical_list: D003451:Cryoprotective Agents; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D005998:Glycine; C100184:tricine; D000432:Methanol