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Low intensity spots of microarray were removed from both channels applying Z-score transformation and we balanced raw intensities applying LOWESS (locally weighted scatter plot smoothing), discarding outlier values from the analysis for every slide.
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After hybridization, the microarray was removed from the MAUI Hybridization Station and immediately immersed in a shallow 250 ml Wash I (Nimblegen, USA) at 42°C for 10 15 s with gentle agitation, then transferred to a second dish of Wash I and incubated for 2 min with gentle agitation.
Two low-quality microarrays were removed from the analysis.
A total of 101 defect arrays and further 801 duplicates of microarrays were removed.
Probe sets declared as present or marginal in less then 80percentt of the analyzed microarrays were removed from the data set.
Two outlier microarrays were removed from each data set (two microarrays from male samples and two from female samples) based on separate hierarchical clustering of each dataset into sample = Cy5 and reference = Cy3 groups.
In the first stage, global dye and microarray effects were removed across all microarray elements or cDNAs transcripts, and in the second-stage individual-study or meta-analyses were implemented.
After quality control, six microarray samples were removed from the original 72 samples and all further analysis were done on the remaining 66 microarray samples.
The log2 intensity values were normalized using a loess transformation, and global dye and microarray effects were removed using the approach implemented Beehive.
Following careful scrutiny of sample identities, all samples that were included in the two previously described pooled node-negative microarray cohorts were removed.
First, all non-specific and partially-specific microarray probe sets were removed from our dataset, leaving only the specific target data, with each probe corresponding typically to only one gene.
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