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With the base-pairing interaction, a quenching probe of two Fc molecules TBA could hybridize with NH2-APs to obtain a low background signal based on the quenching effect of Fc.
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Background signal subtraction was based on wells incubated in the absence of cells.
These background signals were unexpected based on previous results with a radioactive protocol yielding significantly lower background [12].
The signal intensity was background corrected based on the quantile normalization process [ 35, 36] for all microarrays from the entire experiment (under uniform conditions) using data from only perfectly matched oligos.
First, an array-specific background measure, based on the average signal from negative control probes, was subtracted from all probes on the array.
This method has been applied to unambiguously distinguish between the fluorescent protein (GFP) signal in myeloid cells from background autofluorescence based on the fluorescence lifetime.
In addition, we estimated the background "expression" level based on the signal associated with the hygromycin resistance gene from the binary vector pCAMBIA-1305, hph (GenBank accession no. AF354045), included on the array as a negative control and evenly spotted throughout the slides.
In addition, we estimated the background "expression" level based on the signal associated with the hph gene.
RMA starts with a non-linear background correction based on a single chip to detect the perfect match signal.
Raw signal intensities were background corrected using array-specific measures of background intensity based on negative control probes, in the R statistical software (version 2.13) (30) with BioConductor packages (9), before being transformed and normalized using the "vsn" package (12).
To process the signals from the array, quantile normalization (WT vs DKO) and PM-GCBG (signal adjustment based on the background with similar GC content) were applied.
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