BioTechniques最新文献

{"title":"Special approach of droplet digital polymerase chain reaction (ddPCR) for transgene stability of a Chinese hamster ovary (CHO) cell line.","authors":"Shu-Min Zhang, Michele Shannon, Wendy Halsey, Holly Ruess, Christopher Traini, Zhaohui Ao, Xiaodan Ji, Yuan Zhu, Kathleen Van Manen-Brush, Andrew Thomson, Marisa Jones","doi":"10.1080/07366205.2026.2679378","DOIUrl":"10.1080/07366205.2026.2679378","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"239-247"},"PeriodicalIF":2.5,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148210024","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimization of agarose-gelatin double embedding to minimize sectioning artifacts in multicellular spheroids.","authors":"Hyoyoung Maeng, Min-Gi Han, Yoseop Jeon, Donghyeon Kim, Yuna Park, Hyuk Song","doi":"10.1080/07366205.2026.2621053","DOIUrl":"10.1080/07366205.2026.2621053","url":null,"abstract":"<p><p>Processing methods that do not induce shape distortion are essential for the analysis of tissue morphology. Although the use of agarose-gelatin double embedding to reduce shape distortion has been suggested, the effect of matrix concentration has not been addressed. Therefore, combinations of agarose (1-3%) and gelatin (1-10%) were evaluated using multicellular spheroids without extracellular matrix as a model of mechanical fragility. In this study, a blend of 2% agarose and 5% gelatin effectively prevented distortion. This protocol can enhance the morphological analysis of delicate tissues.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"1-5"},"PeriodicalIF":2.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146103645","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Isolation and profiling of single circulating tumor cells in myeloma: a new workflow for liquid biopsies.","authors":"Yulia Shifrin, Asieh Alikhah, Zahabiya Husain, Michelle Nguyen, Atacenk Baslik, Darryl Dyck, Silvana Ferreira, Rayan Kaedbey, Sandra Mazzoni, Sabine Mai","doi":"10.1080/07366205.2026.2645352","DOIUrl":"10.1080/07366205.2026.2645352","url":null,"abstract":"&lt;p&gt;&lt;p&gt;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 10&lt;sup&gt;7&lt;/sup&gt; white blood cells. Isolated cells retain nuclear integrity and cytomorphology, allowing for downstream immuno-phenotyping, three-dimensional (3D) telomere fluorescence &lt;i&gt;in situ&lt;/i&gt; 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&lt;sup&gt;®&lt;/sup&gt; device.Controlled spiking experiments with RPMI 8226 myeloma cells established an analytical limit of detection of approximately 1 tumor cell per 10&lt;sup&gt;7&lt;/sup&gt; 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 &lt;i&gt;in situ&lt;/i&gt; 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 profi","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"123-136"},"PeriodicalIF":2.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147502910","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantitative and unbiased lung alveolar septum assessment in an LPS experimental mouse model using 2D-spatial correlation image analysis from hematoxylin and eosin slides.","authors":"Micaela Lopassio, María José García, Leonel Malacrida","doi":"10.1080/07366205.2026.2674608","DOIUrl":"10.1080/07366205.2026.2674608","url":null,"abstract":"<p><p><b>Introduction:</b> Quantitative assessment of lung tissue architecture is essential for evaluating disease progression in experimental models of acute lung injury (ALI). However, conventional methods for measuring alveolar septum thickness rely on manual procedures that are time-consuming, labor-intensive, and, observer-dependent, compromises comparability across studies and reproducibility in translational biomedical research and drug development.<b>Method:</b> We present a novel, fully automated approach based on two-dimensional spatial autocorrelation function (2D-ACF) analysis to quantify septum thickness from hematoxylin and eosin (H&E)-stained lung sections.</p><p><p>After validation with simulated data, the method was applied to a murine model of ALI induced by lipopolysaccharide (LPS) instillation under normal (ND) or high-fat diet (HFD) conditions.<b>Results:</b> The 2D-ACF provided an unbiased estimate of mean septum thickness across entire tissue images. LPS instillation increased thickness more than sixfold versus controls, with further enlargement in the HFD+Instilled group, capturing additive effects of metabolic stress across >400 images.</p><p><p>Manual measurements showed substantial inter-observer variability, especially in injured lungs, whereas the 2D-ACF was observer-independent and correlated strongly with traditional measurements in the instilled group (<i>r</i> = 0.89, <i>p</i> < 0.001).</p><p><p><b>Discussion:</b> The 2D-ACF framework offers a reproducible, scalable, and unbiased tool for lung pathology studies and other tissues exhibiting spatial heterogeneity in histological architecture.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"189-206"},"PeriodicalIF":2.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148136466","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Antigen retrieval-immunofluorescence on free floating sections to visualize the liver lobule and its cellular makeup.","authors":"Hawon Yoon, Aashita Singh Rajput, Kanesha Lenay Travis, Christopher Michael Glontz, Ekta Pankaj Shah, Parthena Eleni Kotsalidis, Dicle Berfin Azizoglu","doi":"10.1080/07366205.2026.2686546","DOIUrl":"https://doi.org/10.1080/07366205.2026.2686546","url":null,"abstract":"<p><strong>Method summary: </strong>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.</p><p><strong>Multidisciplinary abstract: </strong>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.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"263-270"},"PeriodicalIF":2.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148263200","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"AxioParse: streamlining Axiom Microbiome assay data processing and dataset generation.","authors":"Pranav Kirti, Pirooz Eghtesady, Mathieu Garand","doi":"10.1080/07366205.2026.2644218","DOIUrl":"10.1080/07366205.2026.2644218","url":null,"abstract":"<p><p>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).</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"93-97"},"PeriodicalIF":2.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147472616","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"RP-HPLC-based purification of long single-stranded DNA for CRISPR knock-in applications.","authors":"Justina Martinkienė, Tingting Cui, Giovanni Ciotta, Juozas Kupčinskas, Deividas Pažėraitis","doi":"10.1080/07366205.2026.2649857","DOIUrl":"10.1080/07366205.2026.2649857","url":null,"abstract":"<p><p><b>Background</b>Long 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. <b>Methods</b>We 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. <b>Results</b>The 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. <b>Conclusions</b>This RP-HPLC workflow provides a scalable and reproducible method for generating high-purity long ssDNA suitable for genome engineering applications.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"149-157"},"PeriodicalIF":2.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147637823","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>In vivo</i> RNA targeting in the nematode <i>Caenorhabditis elegans</i> using exogenous catalytic DNA.","authors":"Eriс Khananshvilli, Ayelet-Chen Abraham, Netanel Cohen, Michal Staum, Dorene Friedman, Ithai Rabinowitch","doi":"10.1080/07366205.2026.2678313","DOIUrl":"10.1080/07366205.2026.2678313","url":null,"abstract":"<p><p>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, <i>Caenorhabditis elegans</i>, has not been explored. Here, we examine the feasibility of catalytic DNA-mediated RNA targeting in <i>C. elegans</i>. 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 <i>C. elegans</i>, 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.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"207-223"},"PeriodicalIF":2.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148013277","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correction.","authors":"","doi":"10.1080/07366205.2026.2715298","DOIUrl":"https://doi.org/10.1080/07366205.2026.2715298","url":null,"abstract":"","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"1-2"},"PeriodicalIF":2.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148757736","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Detection of antisense oligonucleotides from biological samples by ligase detection reaction using T4 RNA ligase 2.","authors":"Naoki Harikai, Kohei Kuba, Yuka Ichinose, Akio Hayashi, Yusuke Tanaka, Kazumasa Zaima, Yasunori Uchida, Tokuyuki Yoshida, Hiroshi Sezaki, Takao Inoue","doi":"10.1080/07366205.2026.2716053","DOIUrl":"10.1080/07366205.2026.2716053","url":null,"abstract":"<p><p>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'-<i>O</i>-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.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"293-302"},"PeriodicalIF":2.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148725013","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}