Fermentation time point: 30hr Strain: Lager Yeast Strain CB11
Extracted molecule
total RNA
Extraction protocol
RNA extraction was carried out following the method of Lyne et al. (Lyne, et al., 2003). Cell samples (1x108 cells) were washed with DEPC-treated water, pelleted by centrifugation (13,000 g; 5 minutes) and re-suspended in 750 uL TES. 750 uL of chilled (4°C) acidic phenol:chloroform (5:1; pH 4.7) (Sigma) was added and the samples were incubated on a heating block at 65°C for one hour with agitation of the sample by vortexing every ten minutes for 15 seconds. Samples were then cooled on ice and centrifuged (13,000 g; 4°C; five minutes). The upper phase was transferred to a 2.0 mL Eppendorf Phase Lock GelTM tube (Eppendorf AG, Hamburg, Germany), mixed by inversion and centrifuged (13,000 g; 4°C; five minutes). The upper phase was again removed and transferred to a fresh 2.0 mL Eppendorf Phase Lock GelTM tube containing 700 uL of chloroform:isoamyl alcohol (24:1; Sigma). The samples were mixed by inversion and centrifuged (13,000 g; 4°C; five minutes). The upper phase was transferred to an Eppendorf tube containing 1.5 mL ethanol (-20°C) and 50 uL 3 M sodium acetate pH 5 and agitated briefly by vortexing. RNA was precipitated from the sample by incubation at -70°C for one hour. The sample was then centrifuged (13,000 g; room temperature; five minutes). Supernatant was discarded and the RNA was washed with 70% ethanol (4°C). Ethanol was removed by aspiration and the RNA sample was allowed to dry. 100 uL of DEPC-treated water was added and the RNA sample incubated for ten minutes at room temperature.
Label
biotin
Label protocol
Approximately 5 micrograms of total RNA was reverse-transcribed at 42°C for one hour to generate first-strand cDNA using 100 pmol oligo dT primer containing a 5'-T7 RNA polymerase promoter sequence, 1X Affymetrix First-Strand Master Mix, 10 mM dithiothreitol (DTT), 10 mM dNTPs and 200 units of SuperScript II reverse transcriptase (Invitrogen). Following first-strand synthesis, second-strand cDNA was synthesized using ten units of Escherichia coli polymerase I, ten units of E. coli DNA ligase and two units of RNase H in a reaction containing 1X Affymetrix Second-Strand Master Mix and 10 mM dNTPs. The second-strand synthesis reaction proceeded at 16°C for two hours before ten units of T4 DNA polymerase were added and the reaction allowed to continue for a further five minutes. The reaction was terminated by adding 0.5 M EDTA. Double-stranded cDNA products were purified using the GeneChip® Sample Cleanup Module (Affymetrix). The synthesized cDNAs were in vitro transcribed by T7 RNA polymerase (Enzo BioArray High Yield RNA Transcript Labelling Kit; Enzo Life Sciences Inc., Farmingdale, NY, USA) using biotinylated nucleotides to generate biotinylated complementary RNAs (cRNAs). The cRNAs were purified using the GeneChip® Sample Cleanup Module (Affymetrix). The cRNAs were then randomly fragmented at 94°C for 35 minutes in a buffer containing 40 mM Tris-acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate to generate molecules of approx. 35-200 bp.
Hybridization protocol
= Yeast Genome 2.0 GeneChip® arrays (Affymetrix) were hybridized with 15 micrograms of fragmented, labelled cRNA for 16 hours at 45°C as described in the Affymetrix Technical Analysis Manual. GeneChip® arrays were stained with streptavidin-phycoerythrin solution.
Scan protocol
Samples were scanned using a G2500A GeneArray Scanner (Affymetrix). Following scanning, non-scaled RNA signal intensity (CEL) files were generated using the GeneChip® operating system (GCOS version 5.0; Affymetrix). Non-scaled RNA CEL files contain the raw signal intensity values for each probe on the array, generated from the scanned image of the GeneChip® array. The RNA CEL file contains signal intensity values for the 11 perfect match probes and 11 mismatch probes within each probe set; more than 100,000 signal intensity values for the Yeast Genome 2.0 GeneChip® array.
Description
Yeast sampling: Triplicate samples of the lager yeast strain CB11 were sampled from a 140 hL (14,000 L) propagation vessel immediately after inoculation and at intervals throughout a 30-hour propagation period. The yeast batch had been incubated aerobically in an 8 hL propagation vessel prior to incubation in this principal (140 hL) vessel. Propagation wort (85% malt and 15% sugar adjunct) contained 0.5 mg L-1 Zn2+ (added as zinc sulphate heptahydrate) and was oxygenated with a ramped supply of molecular oxygen (5-100 L min-1) to give a consistent dissolved oxygen concentration of 7-8 mg L-1. Temperature of the wort was maintained throughout at 20°C. Specific gravity of the wort was 17º Plato. Triplicate samples of the same yeast strain were also collected from a fourth-generation 3275hL wort fermentation, i.e. the yeast batch had been used in four fermentations previously. The cylindroconical fermentation vessel was filled in stages, with six successive batches of oxygenated wort (each between 520 and 570 hL) added. The wort used for fermentation was the same as that used for the propagation (85% malt and 15% sugar adjunct), contained 0.2 mg L-1 zinc sulphate and was oxygenated in line prior to vessel filling to achieve a dissolved oxygen concentration of 25 mg L-1. An in line oxygen meter (Orbisphere), upstream of yeast pitching, was used to measure dissolved oxygen. Yeast was pitched with the 2nd and 4th batches of wort. The first sample for analysis was taken 8 hours after all of the yeast had been pitched (and 5 hours after the last batch of oxygenated wort had been added to the fermentation vessel). The fermentation vessel's sample point was located on the vertical side of the vessel, 1 metre above the cone. The sample valves were hygienic needle-type valves. Samples were taken at intervals for up to 102 hours after pitching and were transported between the process vessels and the laboratory at 4ºC and in under 5 minutes. Cells and wort were separated by centrifugation at 4ºC. Cell pellets for RNA extraction were flash-frozen in liquid nitrogen and stored at -80°C until required. Cell density and budding index: Cell suspensions were diluted to an appropriate volume and density was measured using an Improved Neubauer counting chamber (Weber Scientific International Ltd, Middlesex, UK) and standard light microscope (Zeiss, Oberkocken, Germany) at 200x magnification. To determine budding index, a minimum of 500 cells were scored microscopically and the number of budded cells was calculated as a percentage of the total. HPLC analysis of fermentable sugars in wort samples: Wort samples (2 mL) were passed through C18 Plus solid phase extraction cartridges (Waters, Milford, USA) previously conditioned with 5 mL methanol and equilibrated with 5 mL water. The first 1 mL of sample to pass through the cartridge was discarded; the second 1 mL of sample was collected for analysis. An aliquot of the sample (20 µL), was injected onto an amino column (250 x 4.6mm ID, 5µm particle size, Spherisorb NH2, Waters) and the sugars eluted using acetonitrile:water (80:20, v/v) at a flow rate of 3 mL min-1. Detection was achieved using a Wyatt Refractive Index (RI) Detector (Optilab 903, Wyatt Technology Corporation, Santa Barbara, USA). Peak heights were recorded for each compound and concentrations determined by reference to standards of known concentration. The retention times [s] of the sugars were as follows; fructose [185]; glucose [214]; sucrose [338]; maltose [414] and maltotriose [820].
Data processing
Five time points from the fermentation (8, 30, 60, 80 and 102 hours after pitching) were chosen for microarray analysis. All analyses were carried out in triplicate (except for the 30 hour sample from which only duplicate samples were used for analysis), with each replicate processed separately. In total, therefore, 14 samples were used in this investigation. Non-scaled RNA signal intensity (CEL) files were generated using the GeneChip® operating system (GCOS version 5.0; Affymetrix). Non-scaled RNA CEL files contain the raw signal intensity values for each probe on the array, generated from the scanned image of the GeneChip® array. The RNA CEL file contains signal intensity values for the 11 perfect match probes and 11 mismatch probes within each probe set; more than 100,000 signal intensity values for the Yeast Genome 2.0 GeneChip® array.
