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The gene IDs for the genes on the microarray were checked for consistency with the gene annotation presented in AspGD and we found that some of the probes on the microarray were named using the gene alias (alternative gene IDs) in AspGD.
The functionality of the oligos included in the pilot-microarray was checked by hybridizing the same RNA used for the Sanger and 454 sequencing strategies.
The quality of mRNA hybridized to microarrays was checked by RT-PCR experiments on a control (Hprt) and known LIF-induced genes as Socs3, JunB and Fos and the pluripotent Klf4 gene, [ 23, 48],.
The microarrays were checked for quality using the "affy" (version 1.16.0) and " affyPLM" (version 1.14.0) BioConductor packages, 40– 42 as well as an internally developed method (Mahalanobis Distance Quality Control [MDQC], 43 available in the "mdqc" Bionconductor package).
The quality of the microarray experiments was checked by plotting the background of each channel (Cy3 or Cy5) for each microarray and by calculating pair-wise correlations between the signal intensities of each channel of dye-swaps and/or replications (Additional file 3).
Quality of the microarray data was checked using histograms, box plots and a RNA degradation plot.
Then, the consistency of the predictions with microarray data was checked visually in MGcV.
The quality of the microarray data was checked using the 'yaqcaffy' package of the R statistical software (http://www.r-project.org/).org/
In the MAQC project, the quantitative accuracy of several non-microarray devices was checked, then quantitative RT-PCR (Taqman system) was selected as a validation method of microarray data.
Microarray data quality was checked using diagnostic image plots and MA-plots and was found to be satisfactory.
For microarray experiments RNA integrity was checked with the Experion system (Bio-Rad) and samples with a RIN value less than 8 were discarded.
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