Comparison of the transcriptomes of Saccharomyces cerevisiae wild type FY23 and a PDE2 deletion mutant DJ28.
FY23 (Winston et al. 1995) was used as the reference wild-type strain and as the host for the deletion of PDE2 (Guldener et al. 1996; Wach et al. 1994).SFH-PCR mediated replacement (Guldener et al. 1996; Wach et al. 1994) of PDE2 was carried out in FY23 by transforming a KanMX4 cassette amplified from pFA6a-KanMX4. Cells were pelleted by centrifugation, frozen in liquid nitrogen and stored at –80oC until RNA was prepared (Hauser et al. 1998). Frozen cells were transferred to a pre-cooled Teflon vessel containing a 7mm tungsten carbide bead and placed into a Micro-Dismembrator (B. Braun Biotech). Total RNA was extracted using TriZOL reagent (Invitrogen) according to the manufacturer’s instructions. Single-stranded nucleic acids were precipitated with LiCl and resuspended in DEPC-treated water before synthesis and labelling of cDNA (Hegde et al. 2000). Unincorporated fluorescent nucleotides were removed by purification with GFX columns (Amersham Pharmacia). Labelling efficiency was assessed by visualisation on a Storm 860 phosphoimager (Amersham) following agarose gel electrophoresis. Microarrays based on PCR products of all S. cerevisiae open reading frames were manufactured in-house in the Transcriptome Resource Facility of the Consortium for the functional Genomics of Microbial Eukaryotes (COGEME). The differentially labelled samples were hybridised to the array slides overnight at 42oC before being washed as follows: once in 2xSSC, 0.1 % SDS (w/v) for 15min; once in 1xSSC, 0.2% SDS (w/v) for 8min; 0.1xSSC, 0.1% SDS (w/v) for 5min. All washes were carried out in Coplin jars in the dark at room temperature with agitation. The slides were dried by centrifugation at 3000rpm. The hybridised arrays were scanned with a GenePix 4000A scanner (Axon Instruments). Artefacts were removed after visual inspection of the spots and the intensity of fluorescence adjusted to the medians. To minimise the intrinsic variability involved in this technique ten microarray slides were used for the experiment. Half of the slides involved reciprocal dye labelling, allowing compensation for the differences in preferential incorporation of the two fluorescent labels. The labelling will be detailed in the sample. Keywords = PDE2, Ras/cAMP pathway
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