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To confirm the validity of microarray data gene specific mRNAs were quantified from treated and untreated cultures by quantitative real-time PCR (qRT-PCR).
For microarray data, gene expression levels are estimated using the Affymetrix GeneChip and associated manufacturer software.
For the quantitative real time verification of microarray data, gene expression was shown as mean values ± standard deviation (SD).
For pathway analysis of the microarray data, gene lists were analyzed by Ingenuity Pathway Analysis (IPA; Ingenuity® Systems, Redwood City, CA, http://www.ingenuity.com), a web-based bioinformatics tool.
In order to gain insight from the large amount of microarray data, gene expression results were analyzed in the context of biological processes utilizing GeneSpring GX7.3.1 Expression Analysis (Agilent Technologies, USA), Pathway Studio 6 (Adriane Inc).
To assess the validity of the microarray data, gene expression levels for selected chemokines (CXCL1, CXCL3 and CXCL13) were also measured by quantitative real-time polymerase chain reaction (PCR).
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In the microarray data, genes are often represented by multiple oligonucleotide probes.
To analyze the microarray data, genes were grouped based on their expression vectors using the k-means algorithm.
To corroborate the microarray data gene-specific qPCRs were performed.
For the microarray data, genes with fold-change differences in expression were significant after adjustment for multiple testing using the Benjamini-Hochberg method [ 31] (adjusted P ≤ 0.05).
Prior to performing the comparison between RNA-seq and microarray data, genes with more than one predicted isoform were removed from both datasets.
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