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To validate our IL-6 microarray data, relative transcript levels were determined by quantitative real-time PCR.
Validation of microarray data relative to BCL2, EPHA4, EPHA3 and MYF5 expression was performed using both the Universal Probe Library system and sybergreen.
In this case the microarray data relative to deletion line and CS were used.
For the anther microarray data, relative expression levels were extracted from microarray experiments available at NCBI (Gene Expression Omnibus, http://www.ncbi.nlm.nih.gov/geo/[ 72, 73]).
To validate the microarray data, we performed quantitative RT-PCR (qRT-PCR) on several genes that showed increases or decreases in the microarray data relative to the parental RPE1 cells.
Inspection of the microarray data relative to each patient showed that patient 4 with the frameshift mutation c.del58G had reduced RPS19 levels (fold change 0.68) as expected by activation of NMD [ 17, 18].
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For direct comparison with microarray data, the relative changes of mRNA expression were calculated using the ΔΔct method (ΔΔct = Δct sample of interest – Δct control sample) [78].
The results showed that qRT-PCR data was generally consistent with microarray data for relative expression of PtSPLs in roots, stems, young leaves and mature leaves.
Another problem is that microarray data are relative and require incorporation of a control comparison, which may not exist for a field sample.
Two-colour microarray data provide relative measurements of gene expression, and if many samples are analyzed, like in this study, over- and under-representation means in comparison with the whole dataset.
The P-value for the cone enrichment of transcripts that were significantly downregulated (P<0.05) in the microarray data set relative to all detectable transcripts was calculated using one-way ANOVA.
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