2,480 research outputs found
Alien Registration- Bustin, Catherine M. (Portland, Cumberland County)
https://digitalmaine.com/alien_docs/24148/thumbnail.jp
A MIQE-Compliant Real-Time PCR Assay for Aspergillus Detection
PMCID: PMC3393739This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Five years MIQE guidelines: The case of the Arabian countries
The quantitative real time polymerase chain reaction (qPCR) has become a key molecular enabling technology with an immense range of research, clinical, forensic as well as diagnostic applications. Its relatively moderate instrumentation and reagent requirements have led to its adoption by numerous laboratories, including those located in the Arabian world, where qPCR, which targets DNA, and reverse transcription qPCR (RT-qPCR), which targets RNA, are widely used for region-specific biotechnology, agricultural and human genetic studies. However, it has become increasingly apparent that there are significant problems with both the quality of qPCR-based data as well as the transparency of reporting. This realisation led to the publication of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines in 2009 and their more widespread adoption in the last couple of years. An analysis of the performance of biomedical research in the Arabian world between 2001-2005 suggests that the Arabian world is producing fewer biomedical publications of lower quality than other Middle Eastern countries. Hence we have analysed specifically the quality of RT-qPCR-based peer-reviewed papers published since 2009 from Arabian researchers using a bespoke iOS/Android app developed by one of the authors. Our results show that compliance with 15 essential MIQE criteria was low (median of 40%, range 0-93%) and few details on RNA quality controls (22% compliance), assays design (12%), RT strategies (32%), amplification efficiencies (30%) and the normalisation process (3%). These data indicate that one of the reasons for the poor performance of Arabian world biomedical research may be the low standard of any supporting qPCR experiments and identify which aspects of qPCR experiments require significant improvements
Effects of HMGN variants on the cellular transcription profile
High mobility group N (HMGN) is a family of intrinsically disordered nuclear proteins that bind to nucleosomes, alters the structure of chromatin and affects transcription. A major unresolved question is the extent of functional specificity, or redundancy, between the various members of the HMGN protein family. Here, we analyze the transcriptional profile of cells in which the expression of various HMGN proteins has been either deleted or doubled. We find that both up- and downregulation of HMGN expression altered the cellular transcription profile. Most, but not all of the changes were variant specific, suggesting limited redundancy in transcriptional regulation. Analysis of point and swap HMGN mutants revealed that the transcriptional specificity is determined by a unique combination of a functional nucleosome-binding domain and C-terminal domain. Doubling the amount of HMGN had a significantly larger effect on the transcription profile than total deletion, suggesting that the intrinsically disordered structure of HMGN proteins plays an important role in their function. The results reveal an HMGN-variant-specific effect on the fidelity of the cellular transcription profile, indicating that functionally the various HMGN subtypes are not fully redundant
Pitfalls in the normalization of real-time polymerase chain reaction data
Real-time polymerase chain reaction (PCR) is commonly used
for a sensitive and specific quantification of messenger RNA (mRNA). The
levels of mRNA are frequently compared between two or more experimental
groups. However, such comparisons require normalization procedures,
and reference genes are frequently used for this purpose. We discuss pitfalls
in normalization and specifically in the choice of reference genes. Reference
genes, which prove suitable for some experimental conditions, are not necessarily
similarly appropriate for others. Therefore,a proper validation of the
suitability of a given reference gene or sets thereof is required for each experimental
setting. Several computer programmes are available to aid such
validation
Theoretical analysis of the role of chromatin interactions in long-range action of enhancers and insulators
Long-distance regulatory interactions between enhancers and their target
genes are commonplace in higher eukaryotes. Interposed boundaries or insulators
are able to block these long distance regulatory interactions. The mechanistic
basis for insulator activity and how it relates to enhancer
action-at-a-distance remains unclear. Here we explore the idea that topological
loops could simultaneously account for regulatory interactions of distal
enhancers and the insulating activity of boundary elements. We show that while
loop formation is not in itself sufficient to explain action at a distance,
incorporating transient non-specific and moderate attractive interactions
between the chromatin fibers strongly enhances long-distance regulatory
interactions and is sufficient to generate a euchromatin-like state. Under
these same conditions, the subdivision of the loop into two topologically
independent loops by insulators inhibits inter-domain interactions. The
underlying cause of this effect is a suppression of crossings in the contact
map at intermediate distances. Thus our model simultaneously accounts for
regulatory interactions at a distance and the insulator activity of boundary
elements. This unified model of the regulatory roles of chromatin loops makes
several testable predictions that could be confronted with \emph{in vitro}
experiments, as well as genomic chromatin conformation capture and fluorescent
microscopic approaches.Comment: 10 pages, originally submitted to an (undisclosed) journal in May
201
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