More detailed protocols regarding the microarray assay, including microarray preparation, labeling of the cDNA probe, sample hybridization and washing can be obtained from the dbZach website [ 50].
In 25 patients, the same bacteria were identified by microbiological culture and by PCR and microarray assay (including 3 cases where PCR detected only the methicillin-resistance marker gene mecA of staphylococci).
There were 12 additional ORFs that met the 3-fold cutoff for up-regulation by σ in the microarray assay, including genes for pilin biosynthesis (hofB), histidine ammonia lyase (hutH), bEBPs (ygaA, fhlA), propanediol utilization (pduG), siderophore production (iroD), and cell invasion (invG).
Other HrpL up-regulated genes identified in the microarray assay included those with functions related to regulation and chemotaxis, as well as others with unknown function (Table 1; Supplementary Fig. S1).
The tested microarrays were the platforms from Affymetrix, Agilent Technologies, Applied Biosystems, GE Healthcare, Illumina, Eppendorf and the National Cancer Institute, and quantitative gene expression assays included TaqMan® Gene Expression PCR Assay, Standardized (Sta) RT-PCR™ and QuantiGene®.
Given the minimum of biological material required, also attempts have been made to exploit the benefits of microarrays for cellular assays including the detection of MHC-specific T-cell responses.
The genes that are up-regulated in macrophages by hypoxia with a probability of 100% are listed in Table 5. 14 out of 17 genes are known to be up-regulated by hypoxia or they have been validated by independent assays including microarray with other platforms.
On the other hand, we used 6 adult mice and 14 neonate mice from at 4 different litters, which did not include the samples used for the microarray assays, to perform miRNA expression validation assay.
The microarray assays were carried out by Takara Bio. Inc. (Otsu, Japan) using products for microarray analysis manufactured by Agilent Technologies, according to the manufacturer's protocols.
To confirm the differential expression of genes with low expression identified by the SSH/microarray assays (including 446 and 255 up- and down-regulated genes, respectively), we quantified the relative expression level of nine randomly selected genes, whose differential expression was detected by the SSH/microarray assays, in 18 pairs of HCC and the matched non-HCC liver tissues.
