Strategies for simultaneous and successive delivery of RNA

Advanced non-viral gene delivery experiments often require co-delivery of multiple nucleic acids. Therefore, the availability of reliable and robust co-transfection methods and defined selection criteria for their use in, e.g., expression of multimeric proteins or mixed RNA/DNA delivery is of utmost importance. Here, we investigated different co- and successive transfection approaches, with particular focus on in vitro transcribed messenger RNA (IVT-mRNA). Expression levels and patterns of two fluorescent protein reporters were determined, using different IVT-mRNA doses, carriers, and cell types. Quantitative parameters determining the efficiency of co-delivery were analyzed for IVT-mRNAs premixed before nanocarrier formation (integrated co-transfection) and when simultaneously transfecting cells with separately formed nanocarriers (parallel co-transfection), which resulted in a much higher level of expression heterogeneity for the two reporters. Successive delivery of mRNA revealed a lower transfection efficiency in the second transfection round. All these differences proved to be more pronounced for low mRNA doses. Concurrent delivery of siRNA with mRNA also indicated the highest co-transfection efficiency for integrated method. However, the maximum efficacy was shown for successive delivery, due to the kinetically different peak output for the two discretely operating entities. Our findings provide guidance for selection of the co-delivery method best suited to accommodate experimental requirements, highlighting in particular the nucleic acid dose-response dependence on co-delivery on the single-cell level

The efficiency of elemental geochemistry and weathering indices as tracers in aeolian sediment provenance fingerprinting

Confirmation of cost-effective and reliable tracers for aeolian sediment (sand dune) source fingerprinting warrants further research. Accordingly, the main objective of the work reported in this paper was to investigate the efficiency of weathering indices in aeolian sediment fingerprinting using a case study of a fragile arid region in Qom Province, Iran. Eight geochemical elements (Al, Ca, Fe, K, Na, Mg, Si, Ti) and 17 associated weathering indices were measured in 34 aeolian source samples and 10 sand dune target sediment samples in three absolute particulate size fractions. For each fraction, three final composite fingerprints (i.e., geochemical elements only, weathering indices only and a combination of the two) for discerning and ascribing the aeolian sediment sources were selected. The Modified MixSIR Bayesian un-mixing model was used to apportion aeolian source contributions using the final composite fingerprints. Regardless of the composite fingerprint used, all results across the different size fractions suggested that the south-eastern alluvial fan is the dominant (average contribution 50.6%, SD 19.0%) source of the sand dune samples, with the western alluvial fan being the second most important (average contribution 38.4%, SD 20.4%) source. Comparisons of the posterior distributions for the predicted source proportions generated using the nine composite fingerprints (three kinds of composite fingerprints*three particle size fractions) showed that the composite fingerprints combining the geochemical elements and weathering indices generated the most powerful source material discrimination. Our results demonstrate the use of weathering indices alongside more conventional elemental geochemistry tracers for investigations into sand dune sediment provenance