Dense single nucleotide polymorphism (SNP) profiling is central to forensic genetic genealogy (FGG), but challenged forensic samples frequently contain degraded DNA, low quantities of human DNA, and substantial microbial contamination. Although microarrays perform well with high-quality DNA, their performance with challenged forensic specimens is limited. Microarray performance was evaluated on a dilution series of high-quality control DNA and on DNA extracts from 10 skeletal remains that previously yielded successful SNP profiles with massively parallel sequencing (MPS). The skeletal samples represented a range of degradation index values, human DNA quantities, and proportions of human DNA. The high-quality sample series demonstrated robust microarray performance. However, most challenged skeletal samples generated microarray profiles with markedly reduced or inflated heterozygosity, poor signal-to-noise ratios, and large numbers of discordant genotype calls compared with MPS data despite call rates that would traditionally be considered acceptable. Thus, call rate alone can overestimate microarray profile quality with challenged forensic samples; successful microarray genotyping depends on more than human DNA quantity. Because forensic evidence is often limited and irreplaceable, analytical approaches that maximize the likelihood of successful SNP recovery should be prioritized. These findings support MPS as the preferred platform for dense SNP profiling of challenged forensic specimens.
This study evaluated the reduction of selected antibiotic resistance genes (ARGs) in a wastewater treatment plant (WWTP) using conventional activated sludge and assessed the distribution of ARGs in selected Asian communities. Set A samples (n = 21) were collected weekly from May to July 2020 at the influent, secondary-treatment, and effluent stages of a WWTP in Japan, whereas Set B samples (n = 40) were collected between 2022 and 2023 from sites in Japan, the Philippines, Vietnam, and Thailand. Samples were analyzed using a high-throughput quantitative polymerase chain reaction on the Biomark HD System, targeting fecal indicators, mobile genetic element, and ARGs. In Set A, all targeted ARGs and int1 were detected, except for vanA. Substantial ARG concentrations persisted in the effluent. Japan and Thailand samples showed ARG patterns more similar to community-associated resistance, whereas Vietnam and the Philippines samples showed patterns potentially influenced by healthcare-associated sources. Improved treatment and targeted surveillance strategies are necessary for managing the environmental dissemination of antimicrobial resistance.
Previous molecular methods for sex determination of sturgeon of the Acipenser genus have been repeatedly validated for females, but have largely relied on suboptimal solutions to compensate for the lack of a confirmed control for males. By combining sturgeon cytochrome b (cyt b) gene amplification as a reference within existing qPCR techniques, this limitation is addressed through the demonstration of both the presence and confirmed absence of the female-specific AllWSex2 amplicon. Duplexing produces distinct melting curve profiles that enable simultaneous evaluation of both Acipenser confirmation and female identification targets. Incorporating the cyt b primer pair results in clearly distinguishable peak patterns for reliable sex determination, eliminating the need for additional probe detection, and making the approach more cost-effective, faster, and efficient than traditional multiplexing techniques. The method also highlights the importance of developing and appending existing assays to include said endogenous reference controls, such as the cyt b marker used here.
RNA interference (RNAi) screening is widely used for systematic gene perturbation, but reproducible readouts depend on careful assay design and consistent performance. Here, we describe a plate-based small interfering RNA (siRNA) screening workflow that combines repeated green fluorescent protein (GFP) fluorescence measurements (0-72 h) with an orthogonal endpoint XTT metabolic readout performed in the same wells. Using a medium-scale siRNA library and technical replicate plates, we establish a quality control (QC)-guided framework for evaluating assay performance, providing a robust basis for downstream gene-level hit identification. Complementary metrics, including coefficient of variation (CV), strictly standardized mean difference (SSMD), and Spearman rank correlation, were used to assess variability, control separation, and replicate concordance across plates and time points. These analyses revealed improved assay performance at later time points, with increased control separation and reproducibility, supporting the selection of 72 h as the most robust endpoint for downstream comparisons in the present screening workflow. Together, the workflow and QC framework provide a transferable strategy for qualifying plate-based RNAi screening experiments, enabling reliable downstream hit selection and follow-up studies.
This study developed a detection method for antisense oligonucleotides (ASOs) by ligase detection reaction (LDR) using T4 RNA ligase 2 (T4Rnl2). The LDR was applied to detect a nusinersen-based ASO, an 18-mer 2'-O-methoxyethyl-modifiedoligonucleotide with phosphorothioate linkages. The method enabled detection of the ASO at concentrations of 0.08-10 nM in aqueous samples. In addition, when combined with mixed-mode solid phase extraction for ASO spiked samples, the method detected 0.8-100 nM in serum and 0.04-25 nmol/g tissue in spinal tissue. The method was further applied to determine ASO levels in plasma and spinal tissue following intrathecal injection in mice. These results demonstrated that LDR using T4Rnl2 was a simple and sensitive approach for detecting ASOs in biological samples.
