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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.
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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 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],.
All microarray slides were checked for background evenness by viewing the tiff image on Feature Extraction.
Scanned microarray CEL output files were checked for quality assurance and raw signal intensities were RMA normalized using the affy package [61].
The pre-validated breast cancer mRNA biomarkers were checked in other microarray studies that have been conducted in our laboratory on different diseases, including oral cancer [31], primary Sjögren's Syndrome (pSS) [30], pancreatic cancer [34], lung cancer, ovarian cancer, and type 2 diabetes.
RNA samples were checked for quality and integrity with the Agilent 2100 bioanalyzer and used for microarray analysis.
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.
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