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Since gene expression biomarkers typically encompass a range of transcript abundances and differential expression ratios, it is more appropriate to use multiple RNA standards as quality controls for standardizing such measurements, as opposed to a single transcript at a fixed concentration.
We respectively estimated transcript coverage by position and fraction of detected genes in a range of transcript lengths, and found no differences from tube-based single cells and bulk RNA (P = 1, Student's t test, Fig. 2e; P = 1, Student's t test, Additional file 1: Figure S11A).
A range of transcript abundance levels were included in the design of the panels, with the aim of mimicking, for example, transcripts with low abundance in the 'normal' state with a large increase in expression in the 'disease' states (e.g. ERCC-171) or transcripts of average abundance with a moderate fold change in the disease-state (e.g. ERCC-99) (Table 3).
With the aim of mimicking 'normal' and 'disease' states where some biomarkers are differentially expressed whilst others remain unchanged in their expression, standards were spiked at different ratios (1.0, 1.5, 2.0, 5.0, 10.0 and 20-fold differences) over a range of transcript copy numbers (Table 2).
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The RNA tagging conditions were thoroughly optimized and compared to previous methods by using a biochemical oligonucleotide tagging assay and RACE PCRs on a range of transcripts.
The present report focuses on the implementation and validation of the protocol; its sensitivity as compared to previous methods is shown by RACE PCR on a range of transcripts.
RNA editing encompasses a range of transcript-processing mechanisms, including insertions and deletions of single or multiple uridine residues (=U-indel), as well as base modifications and replacements, occurring in both noncoding and protein-coding sequences (Gott and Emeson 2000; Gray 2003).
We show in Drosophila embryos that all mRNAs undergo bidirectional transport on microtubules and that cis-acting elements produce a range of polarized transcript distributions by regulating the frequency, velocity, and duration of minus-end-directed runs.
Other species within subgenus Grammica show a range of rbcL transcript levels, from low to none [ 17], and sampled members of this subgenus lack promoters for plastid-encoded polymerase upstream of the rrn16 and rbcL genes, although transcription of rbcL still occurs from nuclear-encoded polymerase promoter sites in both cases [ 26].
In addition to the more special cases of splicing regulators and snoRNA host genes, the NMD pathway also regulate a range of other transcripts.
For the four libraries developed, our comparative analysis showed that a range of 625 1033 transcripts displayed significant change in expression based on abundance.
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