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The introduction of RNAseq technology further revolutionized the field of gene expression research with accurate measurements of transcripts instead of estimating relative measures and with the detection of structural variants such as splicing and gene fusion.
By averaging the measurements of transcripts corresponding to common genes, we finally obtained the relative expression levels of 15,223 different genes for each expression profile.
Measurements of transcripts that showed strong differences between replicates on average had only half the intensity of truly differentially expressed genes (data not shown).
Additionally, we compare qRT-PCR measurements of transcripts from different ranges of expression intensities to the respective normalized values of the microarray data.
Regarding the quality of the input data, next-generation sequencing technologies (NGST) have overcome some important limitations of microarray technologies, as for example their relatively high rates of false positives and their low accuracy in measurements of transcripts present in low abundance [ 31].
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These measurements of transcript abundance may reflect both changes in gene-specific transcriptional activity as well as changes in cellular composition.
Quantitative measurements of transcript prevalence show that the gene is first activated in the 20-h blastula, but there remain only about 100 molecules of brn1/2/4 mRNA per embryo (only a few per endoderm cell) until an abrupt 10-fold increase occurs as gastrulation begins.
Measurements of transcript level were normalized to rp49.
Comparisons of transcript expression ratios showed greater consistency across platforms than measurements of transcript abundance.
B. Measurements of transcript abundances of hydrogenase-related genes using lysC as an endogenous reference.
Measurements of transcript abundances were performed using the NanoString platform [ 57].
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