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Differential time series gene analysis is previously described [28], MCF10HER2 to MCF10A ratios of gene expression were calculated for each probe at each time point, then normalized to the expression ratio at the zero time point, and finally expressed as the natural logarithm.
For each time point, three arrays were hybridized with RNA derived from one of the PANC-1 cell cultures (technical replicates); the remaining two microarrays were hybridized with RNA from two independent cell cultures (biological replicates), thus generating five data points for each probe at each time point.
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For each probe at least two different tumors per time point were analyzed and gave consisting results.
For each probe (which, at run-time, is continuously delivering information to the controller under test), we apply a single change for each probe data sample.
The raw data from the arrays were analyzed as follows; the log2 ratio (enriched-cy5/total input-cy3) was calculated for each probe and data were then smoothed by averaging across a sliding window of 3 neighbouring probes shifting 1 probe at a time, minimizing noise from single probes.
ISH experiments were carried out three times on n≥5 larvae, for each sense and antisense probe, at each stage.
For dot blots, 1 µL (∼1 µg protein) was spotted on GSWP membranes at each time point and subsequently probed with aptamers.
Probes with a significant time by infection interaction were filtered at each time point (FC ≥ 1.5).
At each time point, ES cell medium was replaced with 100 µl 1 mg/ml MTT (Molecular Probe) for labeling.
The mean hybridization value for each probe set was obtained from the three replicates and was normalized to the mean value for the probe set in the wild-type samples at time zero.
The low signal emitted from the microarray probes can be below the noise level for the chip at each particular time point.
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