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We selected a t-test p-value threshold of 0.1 and a minimum absolute fold difference of 1.4 between the controls and exposed data sets.
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A p-value significance cutoff of 0.05 (without any multiple testing correction) and a minimum absolute fold-change cutoff of 1.2 (typically the lowest sensitivity threshold of commercial microarray platforms) was used to obtain the final set of signatures of differentially-expressed genes.
Of the 719 unique phosphorylated peptides, 24 were down-regulated and 27 were up-regulated with a minimum absolute fold-change of 2.0 and a p-value of less than 0.05 as a function of GRK5 knock-down (Supporting Information, Table S-3).
For further analysis probe sets were filtered by a p-value (t-test) of ≤0.01 and a minimum absolute log2 fold change of 0.2.
We tested four independent ProbeSet-selection techniques: maximum mean signal, maximum absolute fold-change, minimum p-value, and a Probe-to-Gene mapping that remapped all Probes on the array into ProbeSets corresponding to distinct Entrez Gene IDs [ 33].
Model-based t-values that compared treated and vehicle responses on a per time-point basis followed by Empirical Bayes analysis identified 1,206 differentially expressed genes across the time course based on a p1 t) > 0.999 for a minimum of two time points and absolute fold change ≥ 1.5-fold compared to time-matched vehicle control for at least one time point (Fig. 2A).
For this clustering, those profiles that did not show a minimum absolute expression change of 4 log-fold (based on the difference between the minimum and maximum) were filtered out.
From the 42,430 total genes we selected for time-series analysis only those with a minimum absolute expression change between time-points of 4-fold resulting in a total of 2,095 genes.
Minimum absolute per cent error.
Significant regulation at T = 0 was defined as p < 0.05 and an absolute fold change ≥ 2.
FC = fold change, AbsFC = absolute fold change.
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