5-0.6) for a total of three Selleckchem CP673451 sample sets (parallels). Microarrays The microarrays used have been described by Nyquist et al. [32], and a description is available at http://migale.jouy.inra.fr/sakei/Supplement.html/.
70-mer oligonucleotide probes representing the L. sakei strain 23K genome and an additional set of sequenced L. sakei genes were printed in three copies onto epoxy glass slides (Corning). RNA extraction Total RNA extraction was performed using the RNeasy Protect Mini Prep Kit (Qiagen) as described by Rud et al. [33]. The concentration and purity of the total RNA was analysed using NanoDrop ND-1000 (NanoDrop Technologies), and the quality using Agilent 2100 Bioanalyzer (Agilent Technologies). Sample criteria for further use in the transcriptome analysis OICR-9429 research buy were A260/A280 ratio superior to 1.9 and 23S/16S RNA ratio superior to 1.6. cDNA synthesis, labeling, and hybridization cDNA was synthesized and labeled with the Fairplay III Microarray Labeling Kit (Stratagene, Agilent Technologies) as described previously [34]. After labeling, unincorporated dyes were removed from the samples using the QIAQuick PCR purification kit (Qiagen). The following prehybridization, hybridization,
washing, and drying of the arrays were performed in a Tecan HS 400 Pro hybridization station (Tecan) as described by Nyquist et al. [32]. For studying the carbon effects, samples from DMLG and DMLRg were co-hybridized find more for each of the three strains. Separate hybridizations were performed for each strain on all MG-132 three biological parallels. In order to
remove potential biases associated with labelling and subsequent scanning, a replicate hybridization was performed for each strain for one of the three parallels, where the Cy3 and Cy5 dyes (GE Healthcare) used during cDNA synthesis were swapped. The hybridized arrays were scanned at wavelengths 532 nm (Cy3) and 635 nm (Cy5) with a Tecan scanner LS (Tecan). GenePix Pro 6.0 (Molecular Devices) was used for image analysis, and spots were excluded based on slide or morphology abnormalities. Microarray data analysis Downstream analysis was done by the Limma package http://www.bioconductor.org in the R computing environment http://www.r-project.org. Pre-processing and normalization followed a standard procedure using methods described by Smyth & Speed [35], and testing for differential expressed genes were done by using a linear mixed model as described by Smyth [36]. A mixed-model approach was chosen to adequately describe between-array variation and still utilize probe-replicates (three replicates of each probe in each array). An empirical Bayes smoothing of gene-wise variances was conducted according to Smyth et al.
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