Comparison of sampling techniques for parallel analysis of transcript and metabolite levels in Saccharomyces cerevisiae.
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.
Martins
Ana Margarida
AM
Virginia Bioinformatics Institute, Washington Street, Blacksburg, VA 24061-0477, USA.
Sha
Wei
W
Evans
Clive
C
Martino-Catt
Susan
S
Mendes
Pedro
P
Shulaev
Vladimir
V
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*
D003451:Cryoprotective Agents; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D005998:Glycine; C100184:tricine; D000432:Methanol