Hybridization thermodynamics of NimbleGen Microarrays

Background While microarrays are the predominant method for gene expression profiling, probe signal variation is still an area of active research. Probe signal is sequence dependent and affected by probe-target binding strength and the competing formation of probe-probe dimers and secondary structures in probes and targets. Results We demonstrate the benefits of an improved model for microarray hybridization and assess the relative contributions of the probe-target binding strength and the different competing structures. Remarkably, specific and unspecific hybridization were apparently driven by different energetic contributions: For unspecific hybridization, the melting temperature Tm was the best predictor of signal variation. For specific hybridization, however, the effective interaction energy that fully considered competing structures was twice as powerful a predictor of probe signal variation. We show that this was largely due to the effects of secondary structures in the probe and target molecules. The predictive power of the strength of these intramolecular structures was already comparable to that of the melting temperature or the free energy of the probe-target duplex. Conclusions This analysis illustrates the importance of considering both the effects of probe-target binding strength and the different competing structures. For specific hybridization, the secondary structures of probe and target molecules turn out to be at least as important as the probe-target binding strength for an understanding of the observed microarray signal intensities. Besides their relevance for the design of new arrays, our results demonstrate the value of improving thermodynamic models for the read-out and interpretation of microarray signals

Direct amplification results for sample collection strategies including 1000X concentration (20 L hand-filtered to 20 mL) and un-concentrated water, at high initial population abundances (circles; open for concentrated and closed for un-concentrated) and low initial population abundances (triangles; open for concentrated and closed for un-concentrated).

<p>At high abundance, no change in TTP was observed between 1000X concentration and water-only. At low abundance, positive results were observed only after 1000X concentration.</p

MicroRNAs-Based Inter-Domain Communication between the Host and Members of the Gut Microbiome

The gut microbiome is an important modulator of host gene expression, impacting important functions such as the innate immune response. Recent evidence suggests that the inter-domain communication between the gut microbiome and host may in part occur via microRNAs (small, non-coding RNA molecules) which are often differentially expressed in the presence of bacteria and can even be released and taken up by bacteria. The role of microRNAs in microbiome–host communication in intestinal diseases is not fully understood, particularly in diseases impacted by exposure to environmental toxicants. Here, we review the present knowledge in the areas of microbiome and microRNA expression-based communication, microbiome and intestinal disease relationships, and microRNA expression responses to intestinal diseases. We also examine potential links between host microRNA–microbiota communication and exposure to environmental toxicants by reviewing connections between (i) toxicants and microRNA expression, (ii) toxicants and gut diseases, and (iii) toxicants and the gut microbiome. Future multidisciplinary research in this area is needed to uncover these interactions with the potential to impact how gut-microbiome associated diseases [e.g., inflammatory bowel disease (IBD) and many others] are managed

Modeling Hybridization Kinetics of Gene Probes in a DNA Biochip Using FEMLAB

Microfluidic DNA biochips capable of detecting specific DNA sequences are useful in medical diagnostics, drug discovery, food safety monitoring and agriculture. They are used as miniaturized platforms for analysis of nucleic acids-based biomarkers. Binding kinetics between immobilized single stranded DNA on the surface and its complementary strand present in the sample are of interest. To achieve optimal sensitivity with minimum sample size and rapid hybridization, ability to predict the kinetics of hybridization based on the thermodynamic characteristics of the probe is crucial. In this study, a computer aided numerical model for the design and optimization of a flow-through biochip was developed using a finite element technique packaged software tool (FEMLAB; package included in COMSOL Multiphysics) to simulate the transport of DNA through a microfluidic chamber to the reaction surface. The model accounts for fluid flow, convection and diffusion in the channel and on the reaction surface. Concentration, association rate constant, dissociation rate constant, recirculation flow rate, and temperature were key parameters affecting the rate of hybridization. The model predicted the kinetic profile and signal intensities of eighteen 20-mer probes targeting vancomycin resistance genes (VRGs). Predicted signal intensities and hybridization kinetics strongly correlated with experimental data in the biochip (R2 = 0.8131)

Results obtained for different sample types including: <i>Dreissena polymorpha</i> tissues, <i>Dreissena bugensis</i> tissues, <i>D</i>. <i>polymorpha</i> veligers, 1000X concentrated water, and un-concentrated water.

<p>Information for each sample includes the location, the month and year of sample collection, sample processing, primers used, estimated target per reaction, and measured TTP.</p

Isothermal amplification of environmental DNA (eDNA) for direct field-based monitoring and laboratory confirmation of <i>Dreissena</i> sp.

<div><p>Loop-mediated isothermal amplification (LAMP) of aquatic invasive species environmental DNA (AIS eDNA) was used for rapid, sensitive, and specific detection of <i>Dreissena</i> sp. relevant to the Great Lakes (USA) basin. The method was validated for two uses including i) direct amplification of eDNA using a hand filtration system and ii) confirmation of the results after DNA extraction using a conventional thermal cycler run at isothermal temperatures. Direct amplification eliminated the need for DNA extraction and purification and allowed detection of target invasive species in grab or concentrated surface water samples, containing both free DNA as well as larger cells and particulates, such as veligers, eggs, or seeds. The direct amplification method validation was conducted using <i>Dreissena polymorpha</i> and <i>Dreissena bugensis</i> and uses up to 1 L grab water samples for high target abundance (e.g., greater than 10 veligers (larval mussels) per L for <i>Dreissena</i> sp.) or 20 L samples concentrated through 35 μm nylon screens for low target abundance, at less than 10 veligers per liter water. Surface water concentrate samples were collected over a period of three years, mostly from inland lakes in Michigan with the help of a network of volunteers. Field samples collected from 318 surface water locations included i) filtered concentrate for direct amplification validation and ii) 1 L grab water sample for eDNA extraction and confirmation. Though the extraction-based protocol was more sensitive (resulting in more positive detections than direct amplification), direct amplification could be used for rapid screening, allowing for quicker action times. For samples collected between May and August, results of eDNA direct amplification were consistent with known presence/absence of selected invasive species. A cross-platform smartphone application was also developed to disseminate the analyzed results to volunteers. Field tests of the direct amplification protocol using a portable device (Gene-Z) showed the method could be used in the field to obtain results within one hr (from sample to result). Overall, the direct amplification has the potential to simplify the eDNA-based monitoring of multiple aquatic invasive species. Additional studies are warranted to establish quantitative correlation between eDNA copy number, veliger, biomass or organismal abundance in the field.</p></div