Hybridizations were performed overnight under moderate stringency conditions (65°C) in buffer containing 250 mM Na2HPO4 (pH 7.2), 20% SDS, 1 mM EDTA, 0.5% blocking reagent and 50 ng/ml of the probe.
The array design (Agilent SureDesign) had baseline 2× coverage, moderate stringency, moderate masking of repeats, and manual boosting in order to reduce replication of high GC-content baits.
If the database is queried (at moderate stringency, 3.0) for Tribes containing a single gene in each of the seven angiosperm genomes (Arabidopsis, Populus, Vitis, Oryza, Sorghum, Medicago and Carica), this number decreases to 223 genes and if three non-angiosperm plant genomes are added (Selaginella, Physcomitrella, and Chlamydomonas), 36 genes pass the criteria.
These two measures of dispersion were jointly used to detect the outlier transcripts at a moderate stringency level even if the distribution of signal-log ratios may not be normal in a group.
Because the moderate stringency condition (T = 83 °C, Mc = 285 g/kg and Rt = 7 s) completely eliminated detectable S. typhimurium in the test feed matrix, it would appear that all S. typhimurium cells and all mesophilic organisms of similar thermal tolerance would be eliminated at most extruder conditions within the central composite design region.
DIG-labeled probe was hybridized in DIG Easy Hyb buffer (Roche) overnight at 50°C and washed at moderate stringency according to the manufacturer's instructions.
With moderate stringency statistical thresholds (M-value > 0.5 or < −0.5, p-value < 0.05), 9120 genes were differentially expressed: approximately 65% of the genes in the Drosophila genome.
With a moderate stringency screen (RPMK > 3, P < 0.05, FC > 2), there were 355 up-regulated (Additional file 14: Table S14) and 348 down-regulated genes in the Cd2ap mutant endothelial cells (Additional file 15: Table S15).
For instance, at moderate stringency (TST-adjusted p ≤ 0.01), 25,923 genes (91.4% of all detectable genes) showed significant expression differences among the three soybean lines.
Similarly, the PPR-like 1 gene aligns to a portion of PPR-like 2. All of these alignments were using the "moderate" stringency function of GeneSeqer.
