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The microarray did not show complete loss of STMN1 in the STMN-/ MEFs because the probe design for STMN1 is flawed.
Especially, because the probe design for a gene is based on the exons included in one or multiple manually annotated or computationally predicted gene models [ 5], many infrequently used exons are never represented by any probe, and therefore their expression levels cannot be measured accordingly.
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Thirteen gene groups containing a total of 14 sequences were excluded from the probe design because of redundancy.
Genome sequences for these were not included in the probe design because they became available only after we designed the microarray probes or because they were not classified into a bacterial taxonomic family; therefore probes were not screened for cross-hybridization against these targets.
The probe design is simple because it consists solely of the elimination of guanine paired with the target cytosine from a full match sequence.
Details of the probe design have been described previously [28].
Of the 44 132 sequences used as input in the probe design pipeline, 2 021 transcripts were discarded either due to presence of overlapping probes and possible cross hybridization, or because no satisfactory probe design was possible.
The probe design is available online [ 39].
For further testing in humans, we favour the probe QE; because of the applied probe design strategies, the introduction of negatively charged d-glutamic acids favoured probe characteristics more than multimerization of the molecule.
The probes designed for most of the module 2 genes seem to show unreliable expression because the probes of 33 out of the 40 genes were designed based on fragment sequences of full-length cDNA clones.
Because the probe is designed to selectively bind to the intended template only, fluorescent signals decline in case of non-specific amplification.
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