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For the first simulation, we selected γ ig values whose mean for the patients is lower than that for the controls by an amount that counter balances the average difference in mean expression between patients and controls for truly differentially expressed genes.
We also calculated the difference in mean expression between the fields for each season.
We performed unpaired two-sample t-tests in GENESPRING 7 to find genes with different mean expression between disease groups.
Values with a p-value < 0.05 were extracted and sorted by the difference in mean expression between the two groups.
For each gene the difference in mean expression between the two temperatures is a proxy for the phenotypic plasticity of this gene's expression.
We based a priori gene selection for Ingenuity Pathway Analysis (IPA®) on a significant difference in mean expression between the two groups of 5-fold.
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Correlations between mean expression values between groups were calculated.
To shortlist subset miRs regulated consistently in terms of biological and technical stability with more stringency for further analysis, we considered those miRNAs for which the distance between mean expression values between tumour-bearing and sham mice was at least five times greater than the sum of standard deviation of two groups.
Genes were considered to be differentially expressed if the absolute fold-change (Fc) in mean expression values between D1-GFP-expressing cells and GFP-expressing cells was at least 1.5.
Of the p genes, m are assumed differentially expressed with difference in mean expression levels between classes of 2 δ and the remaining p-m genes are not differentially expressed.
Distinct thresholds of Q-values, pairwise comparisons, and relative differences of mean expression levels between both lines (fold change, FC) were combined to define differentially expressed probe sets, respectively transcripts (see Results).
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