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Further scaling the data to a common reference sample (herein referred to as total count scaling) introduces more variability by pushing all samples toward the same distribution, especially for samples with different sequencing depth.
A common reference sample was constructed from a pool of all labeled RNA extracts.
Allele size lengths have been standardized via comparison with a common reference sample.
In each measurement, individual samples were measured together with a common reference sample comprised of all 12 samples used in this study.
As cluster analysis was the major goal of this study the microarray experiment followed a reference design, where all samples were hybridized against a common reference sample.
Protein expression was calculated as a ratio of the protein expression level in a pooled sample to the protein expression level in a common reference sample comprised of all 12 samples used in this study.
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Samples were co-hybridized against a whole-newborn mouse total RNA as common reference sample using a replicated dye-swap design.
Microarrays were competitively hybridized according to Agilent specifications for two-color gene expression at 65 °C for 17 h, each tissue hybridized against the common reference sample for a total of eight microarrays.
Microarray hybridizations were performed using Agilent Human oligonucleotide (1Av1, 1Av2 and custom designed 1Av1-based) microarrays using 2 μg of Cy3-labeled common reference sample that is a modified version of the Stratagene Human Universal Reference[ 36], and 2 μg of Cy5-labeled experimental sample.
To detect differentially expressed genes we used a linear model that compared all treatments against the common reference sample.
In addition, the same common reference sample has been used in a separate microarray experiment investigating the response of bovine monocytes to T. annulata infection (Jensen et al., in prep).
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