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.
In laboratory mice, the H2-Ld gene confers resistance to encephalitis upon infection with Toxoplasma gondii, a foodborne parasite capable of infecting all endotherm animals tested. H2-Ld is carried by inbred strains with major histocompatibility complex (MHC) haplotypes H-2a (e.g., A/J) and H-2d (e.g., BALB/c), but not by those with haplotypes H-2b (e.g., C57BL/6) and H-2k (e.g., C3H/He). As H2-Ld is typically detected by serological typing of ex vivo spleen samples, testing is usually terminal. Here we present a polymerase chain reaction (PCR)-based method to reliably detect the H2-Ld gene in ear biopsies from live mice, thus allowing researchers to ascertain the H2-Ld genotype for breeding or other in vivo procedures.
This protocol describes a methodology combining TurboID - a recently developed proximity biotinylation technique - with conventional epitope-tag based co-immunoprecipitation (Co-IP) to analyze protein-protein interactions (PPIs) in cell culture systems. This integrated approach allows the targeted examination of both transient and stable protein interactors, enhancing our understanding of protein dynamics. TurboID captures transient interactions often missed by Co-IP, which captures high affinity, stable interactors. Combination of both techniques enables direct comparison of interaction strengths, providing insights into the dynamic nature of protein interactions within cells. The rapid biotinylation capability of TurboID reduces background noise and false positives while Co-IP enriches stable interactors, together improving data quality and interpretation of the interactomic landscape. Demonstrating the efficacy of this methodology, proteins relevant to the pathology of Spinal Muscular Atrophy were utilized to explore variations at the interactome level. The use of identical starting cell lysates for both TurboID and Co-IP minimized variability and ensured datasets were comparable, allowing for consistency and enhancing the reliability of findings regarding the nature and strength of protein interactions. This novel framework effectively combines both innovative and classical techniques while maintaining consistency in sample handling, advancing our understanding of the intricate networks that govern cellular processes. Full data-set available at https://www.ebi.ac.uk/pride/ reference ID PXD066858.
METHOD SUMMARY:Paraformaldehyde-fixed mouse liver samples are sectioned on a vibratome at 100 µm. The free-floating sections are permeabilized, followed by exposure to one of two heat-induced antigen retrieval methods selected based on the antigen of interest. Antigen retrieval is carried out in a mechanical stabilization apparatus developed here to prevent free-floating sections from deforming. The immunostaining is then carried out with noted modifications to previously established protocols. MULTIDISCIPLINARY ABSTRACT:Spatial organ biology widely relies on slicing tissues into one cell-thick sections. These studies have advanced our understanding of organ structure and function; yet, thin sections have been insufficient to investigate three-dimensional organization in complex organs. A more recent approach utilizes thicker sections, enabling visualization of volumetric tissue architecture. Currently, few protocols exist to strengthen immunofluorescence signal on thick tissue sections while maintaining tissue integrity, particularly for difficult antigens. Here, we developed a simple, affordable method that mechanically stabilizes thick tissue sections and preserves tissue integrity during heat-mediated antigen retrieval used to enhance antigen accessibility. This method widens the utility of thick tissue sections and facilitates holistic analysis of complex tissues, opening new possibilities for spatial organ research.
Cell line stability of mammalian cell lines is an important concern for manufacturing of biopharmaceuticals. Characterization of the master cell bank (MCB) for monoclonal antibody A (Mab A) via multiplex fluorescence in situ hybridization (M-FISH) analysis identified two distinct karyotypic cell populations. As a result, a regulatory agency requested additional analysis to support a single clonal lineage of the MCB and demonstrate control of the two cell populations in subsequently derived cell banks. Next Generation Sequencing (NGS) revealed 11 transgene integration site sequences. A multiplex (measure two amplicons) Droplet Digital PCR (ddPCR) method was developed and characterized those 11 integration site copy numbers in the MCB. Analysis of 12 subclones from the MCB displayed that among 7 subclones there was loss of at least one of five specific integration sites. The characterization of these five unstable sites in the end production cells (EPCs) of four manufacturing batches are consistent with the MCB. The conservation of five other integration sites among all clones supported that these cells were derived from the same population and that loss of the unstable integration sites contributed to the two distinct karyotypic populations.
Long interspersed nuclear element-1 (LINE-1 or L1) is the only autonomously active retrotransposon in the human genome and produces both sense and antisense transcripts from its 5' untranslated region (5'UTR) of L1Hs, a human-specific L1 subfamily. Among these, ORF0 is an antisense transcript-derived protein implicated in retrotransposition activity, yet its mRNA expression has been difficult to quantify because strand discrimination is required for accurate detection. Here, we developed a strand-specific quantitative polymerase chain reaction (qPCR) method incorporating multiplex gene-specific tagged primers in the reverse transcription (RT) step, which enables simultaneous quantification of antisense ORF0 and sense 5'UTR and ORF2 transcripts. Validation in 5-aza-2'-deoxycytidine-treated neuroblastoma cells confirmed dose-dependent increases in expression of ORF0 and sense transcripts, demonstrating the strand specificity and functionality of this method. Application to human postmortem prefrontal cortex and cerebellum samples revealed stable expression of ORF0 and sense transcripts. This assay provides a robust and scalable tool for the precise quantification of strand-specific L1 transcription, complementing locus-specific analyses, and offering a platform for future studies on the role of ORF0 and L1 antisense transcription.
Catalytic DNA molecules (DNAzymes) offer a programmable means to manipulate RNA without genomic modification. Although DNAzymes have been thoroughly characterized in culture and in various animals, their utility in the prevalent model system, Caenorhabditis elegans, has not been explored. Here, we examine the feasibility of catalytic DNA-mediated RNA targeting in C. elegans. Cholesterol-conjugated 8-17 DNAzymes were efficiently delivered to intestinal cells following ingestion, with no visibile toxic effects on growth or health, reducing targeted reporter fluorescence and endogenous gene function. The observed dependence on cholesterol tagging was consistent with a requirement for cellular uptake. Our findings provide a proof-of-principle demonstration of catalytic DNA activity in C. elegans, and highlight considerations for design and optimization. This exploratory study lays the foundation for expanding catalytic nucleic acid technologies in nematodes, supporting future development of programmable RNA tools in a powerful model system.
Antibiotic resistance genes (ARGs) are important contaminants in water systems, and their detection depends strongly on methodological sensitivity. This study compared three molecular platforms, high-throughput quantitative polymerase chain reaction (HT-qPCR), hydrolysis probe-based qPCR, and droplet digital PCR (ddPCR), for detecting ARGs in wastewater, river water, and seawater in Thailand. HT-qPCR enabled broad resistome profiling, detecting 325-336 ARGs out of 373 targets (87.1-90.1%), with aminoglycoside, beta-lactam, macrolide-lincosamide-streptogramin B, sulfonamide, mobile genetic element, and integron genes most prevalent. qPCR quantified selected ARGs using standard curves constructed from plasmid standards whose absolute copy numbers were calibrated by ddPCR, yielding high efficiency and strong linearity. ddPCR detected the same target genes as qPCR with comparable concentration estimates and additionally identified blaIND, which was not observed by HT-qPCR or qPCR. Quantifications from qPCR and ddPCR showed high rank-based concordance across matrices. Combining HT-qPCR with qPCR improved coverage by about 1% relative to HT-qPCR alone, while HT-qPCR with ddPCR offered nearly identical values. In conclusion, HT-qPCR was most effective for comprehensive profiling, qPCR ensured reliable quantification, and ddPCR enabled detection of rare targets, supporting a tiered and cost-effective framework for smart ARG monitoring in tropical aquatic environments.
The presence of 5-methylcytosine at CpG sites in mammalian DNA plays a significant role in various biological processes, both normal and aberrant. Similarly, CpG site methylation status of therapeutic DNAs can affect activity and therapeutic efficacy, as is the case for ADI-100, an immune tolerance-inducing drug candidate for the treatment of type 1 diabetes and other GAD-associated autoimmune diseases. To assess the methylation characteristics of ADI-100 and other therapeutic DNA product candidates, we developed a simple and versatile method, methylation-sensitive restriction enzyme digest and agarose gel electrophoresis (MSRE-AGE), using methylation-sensitive restriction enzyme digest, agarose gel electrophoresis, and custom in silico band pattern analysis for quantitative measurement of CpG methylation levels and patterns. We compared MSRE-AGE with bisulfite pyrosequencing methylation analysis of plasmid DNA and a synthetically-produced closed-linear DNA, Doggybone DNA (dbDNA), demonstrating that MSRE-AGE produces results consistent with bisulfite pyrosequencing but without structure and sequence-dependent artifacts. These findings demonstrate that MSRE-AGE is a robust and practical method for methylation analysis of therapeutic DNA products.
BackgroundLong single-stranded DNA (ssDNA; >200 nucleotides) is valuable for DNA nanotechnology, precision medicine, and as a CRISPR-Cas9 knock-in donor template, but existing preparation methods are laborious, low-yield, or difficult to scale. MethodsWe developed a workflow combining enzymatic digestion with high-temperature reversed-phase high-performance liquid chromatography (RP-HPLC) to purify kilobase-length ssDNA. The method was evaluated across analytical and semi-preparative formats. ResultsThe approach enables clean resolution of linear and circular ssDNA species ranging from 1.5 to 4.5 kb and is scalable across formats. A 1.5 kb ssDNA donor supported efficient CRISPR knock-in at the T-cell receptor alpha constant (TRAC) locus in primary human CD8⁺ T cells, without adversely affecting viability or expansion. ConclusionsThis RP-HPLC workflow provides a scalable and reproducible method for generating high-purity long ssDNA suitable for genome engineering applications.
Introduction: Low-density lipoprotein receptor-related protein 8 (LRP8) is a neuronal receptor for apolipoprotein E and Reelin, two ligands critically involved in Alzheimer’s disease (AD), neuronal migration, and memory. Because LRP8 is highly expressed in neurons, interacts with amyloid precursor protein, and undergoes γ-secretase-dependent processing, it has emerged as a potential contributor to AD-related neurodegeneration. Growing evidence also implicates LRP8 in carcinogenesis, highlighting the need to better define its molecular properties. Areas covered: This article addresses the limited understanding of LRP8 proteolytic processing, cellular localization, and molecular interactions, due in part to the lack of suitable antibodies. We present and characterize novel polyclonal and monoclonal antibodies directed against the C-terminal region of LRP8, suitable for Western blotting and immunocytochemistry/immunofluorescence. These reagents enabled detection of a previously unrecognized intracellular low-molecular-weight (∼12 kDa) C-terminal LRP8 fragment. Expert opinion/Commentary: These antibodies provide valuable new tools for mechanistic studies of LRP8. By improving the investigation of LRP8 processing and localization, they may facilitate a better understanding of its role in neurodegeneration and cancer.
Introduction.- Affordable and accessible Deoxyribonucleic Acids (DNA) detection remains a major challenge in global healthcare because most molecular diagnostic techniques rely on chemical labels, complex reagents, and expensive equipment. Objective.- In this study, we investigate a fundamentally different approach based on Magnetic Induction Spectroscopy (MIS) to detect DNA concentrations in solution without labels, reagents, or physical contact. Methods.- Bulk electrical propertiess of DNA at multiple concentrations informed a three-dimensional electromagnetic simulation model. Results.- Multifrequency analysis revealed clear differences in the inductive phase shift as a function of DNA concentration, with maximum sensitivity near 1 MHz. Conclusions.- Simulated and extrapolated results indicate a potential detection range of 0.06-1000 μMol, encompassing typical post-PCR concentrations. The findings demonstrate modeling-based feasibility for concentration-dependent detection of bulk DNA.
Mitochondria, with their own DNA, Represent a potential target for nucleic acid-based precision therapies. However, effective delivery of therapeutic oligonucleotides remains challenging due to the dual mitochondrial membranes and the localization of mitochondrial DNA within nucleoid complexes in the matrix. To understand the delivery process and assess the delivery efficiency of potential vectors, such as dendrimers, it is essential to effectively quantify the oligonucleotides that are successfully delivered to and remain within mitochondria. Currently, there are only limited yet inconvenient methods available for this purpose. Here, we describe a method for quantifying the delivery of fluorescent oligonucleotide cargos in isolated mitochondria using a microfiltration apparatus for reliable fluorescent analysis. By working within a range of dilutions, we are able to safeguard the concentration limits. The quantification protocol also enables the visualization of specific localization within mitochondria, allowing for the determination of whether delivery can occur across both membranes. This is particularly useful, as it offers a key insight into improving vectors as they must deliver the cargoes within the mitochondrial matrix. We validate this method in this proof-of-concept study, providing biological data to assess the difference between two amphiphilic dendrimer vectors for oligonucleotide delivery in mitochondria.
Minimal residual disease (MRD) is a key prognostic marker for progression-free and overall survival in multiple myeloma (MM). Existing high sensitivity assays primarily focus on tumor burden assessment, rely on bone marrow sampling, and are limited in their ability to support frequent longitudinal disease monitoring. Here, we describe a proof-of-principle workflow for isolating morphologically preserved circulating tumor cells (CTCs) from peripheral blood (PB) using size-based filtration. Based on controlled spiking experiments with RPMI 8226 myeloma cells, we demonstrate an analytical limit of detection of approximately 1 tumor cell per 107 white blood cells. Isolated cells retain nuclear integrity and cytomorphology, allowing for downstream immuno-phenotyping, three-dimensional (3D) telomere fluorescence in situ hybridization (FISH), and single-cell telomere profiling, a known marker of genomic instability and disease progression in multiple myeloma. The proposed workflow demonstrated its feasibility for isolating, profiling, and analyzing plasma cells from PB of MM patients at different disease stages. It revealed distinct nuclear and telomeric features in MM CTCs compared with normal lymphocytes. The established technically robust liquid biopsy workflow enables 3D telomere profiling of MM CTCs that can be adopted for noninvasive MRD monitoring based on genomic instability rather than on the enumeration of MM plasma cells alone.Article HighlightsCurrent high-sensitivity assays for assessing minimal residual disease (MRD) in multiple myeloma (MM) patients rely on invasive bone marrow sampling and are limited by sampling bias and poor suitability for frequent longitudinal monitoring.This study presents a proof-of-principle liquid biopsy workflow that enables isolation of morphologically intact circulating tumor cells (CTCs) from peripheral blood (PB) using size-based filtration with the ScreenCell® device.Controlled spiking experiments with RPMI 8226 myeloma cells established an analytical limit of detection of approximately 1 tumor cell per 107 white blood cells.Technical feasibility of the new workflow for isolating intact CTCs from liquid biopsy was confirmed in a cohort of 20 newly diagnosed MM patients at diagnosis, during induction therapy, and after relapse, supporting its potential utility for longitudinal disease monitoring.Isolated CTCs were successfully immunophenotyped and subjected to quantitative three-dimensional telomere fluorescence in situ hybridization (FISH), allowing single-cell analysis of telomere length, number, aggregation, nuclear volume, and spatial distribution.Quantitative telomere profiling revealed statistically significant differences in nuclear and telomeric parameters between MM CTCs and normal lymphocytes, consistent with known markers of genomic instability and disease aggressiveness in MM.By combining enumeration with risk assessment based on telomere profiling, the current workflow can provide clinicians with much-needed biological insight beyond mere tumor burden assessment. Incorporating minimally invasive telomere profile-based risk assessment into MRD guidelines may guide treatment decisions in cases of sustained MRD and inform the need for new treatment regimens when residual disease is detected.
The Applied Biosystems Axiom Microbiome Array enables high-throughput detection of bacteria, archaea, viruses, protozoa, and fungi across multiple samples. However, its native software outputs are not compatible with common downstream analysis tools, requiring preprocessing. We identified a lack of open-source pipelines tailored to these outputs. To address this gap, we developed AxioParse, a Python-based pipeline built with the Dagster orchestration framework that automates data cleaning, taxonomic mapping, and formatting for downstream analysis. AxioParse reduces manual processing and generates datasets compatible with platforms such as QIIME2 and R, improving reproducibility and facilitating broader use of the Axiom Microbiome Array in microbiome research (https://github.com/Eghtesady-Lab-Bioinformatics/axioparse).
