Analysis of clinical trial specimens such as formalin-fixed paraffin-embedded (FFPE) tissue for molecular mechanisms of disease progression or drug response is often challenging and limited to a few markers at a time. This has led to the increasing importance of highly multiplexed assays that enable profiling of many biomarkers within a single assay. Methods for gene expression analysis have undergone major advances in biomedical research, but obtaining a robust dataset from low-quality RNA samples, such as those isolated from FFPE tissue, remains a challenge. Here, we provide a detailed evaluation of the NanoString Technologies nCounter platform, which provides a direct digital readout of up to 800 mRNA targets simultaneously. We tested this system by examining a broad set of human clinical tissues for a range of technical variables, including sensitivity and limit of detection to varying RNA quantity and quality, reagent performance over time, variability between instruments, the impact of the number of fields of view sampled, and differences between probe sequence locations and overlapping genes across CodeSets. This study demonstrates that Nanostring offers several key advantages, including sensitivity, reproducibility, technical robustness, and utility for clinical application.
Changing precipitation patterns are predicted to alter ecosystem structure and function with potential carbon cycle feedbacks to climate change. Influenced by both land and sea, salt marshes are unique ecosystems and their productivity and respiration responses to precipitation change differ from those observed in terrestrial ecosystems. How salt marsh greenhouse gas fluxes and sediment microbial communities will respond to climate‐induced precipitation changes is largely unknown. We conducted 1‐year precipitation manipulation experiments in the Spartina patens (high marsh) zone of two salt marshes and quantified ecosystem functions at both and microbial community structure at one. Precipitation treatments (doubled rainfall, extreme drought, and seasonal intensification) had a significant, although transient, impact on porewater salinity following storms at both sites, but most site conditions (nutrient concentrations, sediment moisture, and temperature) were unaffected. Extreme drought led to a subtle change in microbial community structure, but most ecosystem functions (primary productivity, litter decomposition, and greenhouse gas fluxes) were not affected by precipitation changes. The absence of ecosystem function change indicates functional redundancy (under extreme drought) and resistance (under doubled precipitation and seasonal intensification) exist in the microbial community. Our findings demonstrate that salt marsh ecosystems can maintain function (including ecosystem services like carbon sequestration) under even the most extreme precipitation change scenarios, due to resistance, resilience, and functional redundancy in the underlying microbial community.
Ocean acidification is predicted to impact the nitrogen cycle in a variety of ways. Specifically, manipulations of water column pH have shown that nitrification, the microbial conversion of ammonium to nitrate, is inhibited at low pH. A decrease in nitrification may impact phytoplankton composition and production, denitrification, and the production of nitrous oxide. We compiled an existing unique data set of concurrent water column nitrification rates and water column pH values from a temperate New England estuary (Narragansett Bay, RI, USA). Contrary to the current hypothesis, we found that nitrification rates were highest at low pH and significantly (P=0.0031) lower at high water column pH. In this study, pH varied up to 0.85 units, 20% more than the maximum predicted ocean pH decrease of 0.7 units. These results highlight that nitrifying organisms in coastal systems tolerate a wide range of pH values. Moreover, the degree of negative correlation with pH may depend on site-specific environmental conditions. Combined, these findings indicate that the current hypothesis of the negative impacts of ocean acidification on nitrification, at least for the coastal ocean, might need reevaluation.
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