BioTechniques最新文献_第3页

Isolation and characterization of bovine-milk derived extracellular vesicles using a modified aqueous two-phase system. 用改进的双水相系统分离和表征牛奶来源的细胞外囊泡。

IF 2.6 4区工程技术

BioTechniques Pub Date : 2026-01-01 Epub Date: 2026-03-17 DOI: 10.1080/07366205.2026.2633107

Logan Scott Whitney, Elley Ruth College, Jonah Peña-Ekker, Charlee Ann Cannon, Kolbe Mark Mason, Jaren Nathan Wilson, Jessica E Pullan

{"title":"Isolation and characterization of bovine-milk derived extracellular vesicles using a modified aqueous two-phase system.","authors":"Logan Scott Whitney, Elley Ruth College, Jonah Peña-Ekker, Charlee Ann Cannon, Kolbe Mark Mason, Jaren Nathan Wilson, Jessica E Pullan","doi":"10.1080/07366205.2026.2633107","DOIUrl":"10.1080/07366205.2026.2633107","url":null,"abstract":"Extracellular vesicles (EVs) are lipid bilayer-bound nanoparticles secreted by nearly all cells, with notable applications in intercellular communication and therapeutic delivery. However, conventional EV isolation techniques, such as ultracentrifugation and size exclusion chromatography, often face challenges like high cost, specialized equipment requirements, and low yield. In this study, we present a modified aqueous two-phase system (ATPS) as an alternative method for isolating EVs from raw bovine milk. This approach is scalable, cost-effective, and requires minimal specialized equipment, making it accessible for small laboratories. We demonstrate the efficiency of this method through the isolation and subsequent characterization of bovine milk-derived EVs, including morphological analysis, protein, and lipid quantification, and nucleic acid presence. The isolated EVs exhibited typical characteristics, morphology, specific protein markers (CD63 and TSG101), and a protein-to-lipid ratio consistent with extracellular vesicles. These findings validate the modified ATPS as a reliable and practical method for isolating milk-derived EVs, offering a promising tool for future research into their therapeutic potential and other applications.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"55-64"},"PeriodicalIF":2.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147497751","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}

引用次数: 0

A PCR method to detect the mouse major histocompatibility complex H2-L^d gene that confers resistance to Toxoplasma encephalitis. 采用PCR方法检测小鼠抗弓形虫脑炎主要组织相容性复合体H2-Ld基因。

IF 2.6 4区工程技术

BioTechniques Pub Date : 2026-01-01 Epub Date: 2026-08-01 DOI: 10.1080/07366205.2026.2709365

Natalie E Bicknell, Aseel Abuzaid, Glenn A McConkey, Steven J Clapcote

引用次数: 0

Optimized membrane filtration for high-purity red blood cell isolation from human and murine samples. 优化了从人和鼠样品中分离高纯度红细胞的膜过滤。

IF 2.6 4区工程技术

BioTechniques Pub Date : 2026-01-01 Epub Date: 2026-07-20 DOI: 10.1080/07366205.2026.2699085

Luis E F Almeida, Meghann L Smith, Sayuri Kamimura, Kamille West-Mitchell, Zenaide M N Quezado

引用次数: 0

Optimized Southern blotting for enhanced and precise detection of transgenes in CHO cells from transposon-based expression systems. 优化的Southern印迹法用于从转座子表达系统中增强和精确检测CHO细胞中的转基因。

IF 2.6 4区工程技术

BioTechniques Pub Date : 2026-01-01 Epub Date: 2026-01-29 DOI: 10.1080/07366205.2026.2621051

Hyo-Young Jeong, Caitlyn Devine, Bor-Ruei Lin, Zhenqiu Huang, Guanghua Li, Lin Zhang

{"title":"Optimized Southern blotting for enhanced and precise detection of transgenes in CHO cells from transposon-based expression systems.","authors":"Hyo-Young Jeong, Caitlyn Devine, Bor-Ruei Lin, Zhenqiu Huang, Guanghua Li, Lin Zhang","doi":"10.1080/07366205.2026.2621051","DOIUrl":"10.1080/07366205.2026.2621051","url":null,"abstract":"The genetic stability of recombinant CHO cell lines producing therapeutic proteins is critical for ensuring consistent quality in biopharma-ceutical products. Southern blotting remains the gold standard for evaluating transgene integrity and stability in these cell lines. In the biopharmaceutical industry, transposon-based expression systems are widely utilized to generate highly productive and genetically stable CHO cell lines. However, evaluating transgene integration sites and integrity in such cell lines is challenging with standard Southern blotting protocols. This difficulty arises because transposon-mediated transfection often results in multiple independent integration sites in the host genome, each typically harboring a single transgene copy. Upon restriction enzyme digestion, similar-sized DNA fragments are generated, reducing resolution and complicating the separation and detection of the transgenes using standard blotting protocols. Here, we present a modified Southern blotting protocol that significantly improves the resolution of integration banding patterns by refining key steps, including purification of digested DNA prior to electrophoresis and an enhanced DNA transfer method. This protocol was successfully applied to analyze multiple transposon-derived CHO cell lines with high transgene copy numbers, enabling more precise and efficient detection of transgene integration.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"1-10"},"PeriodicalIF":2.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146084041","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}

引用次数: 0

A QC-guided framework for performance assessment in plate-based RNAi screening with fluorescence and metabolic readouts. 基于荧光和代谢读数的板基RNAi筛选性能评估的qc指导框架。

IF 2.6 4区工程技术

BioTechniques Pub Date : 2026-01-01 Epub Date: 2026-08-30 DOI: 10.1080/07366205.2026.2723761

Leonie Stahl, Anja K Bosserhoff

{"title":"A QC-guided framework for performance assessment in plate-based RNAi screening with fluorescence and metabolic readouts.","authors":"Leonie Stahl, Anja K Bosserhoff","doi":"10.1080/07366205.2026.2723761","DOIUrl":"https://doi.org/10.1080/07366205.2026.2723761","url":null,"abstract":"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.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"311-321"},"PeriodicalIF":2.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148860457","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}

引用次数: 0

Quantitative analysis of oligonucleotide delivery into isolated mitochondria: a proof-of-concept method. 寡核苷酸递送到分离线粒体的定量分析：一种概念验证方法。

IF 2.6 4区工程技术

BioTechniques Pub Date : 2026-01-01 Epub Date: 2026-03-26 DOI: 10.1080/07366205.2026.2635461

Joshua McHale, Veronica Bazzani, Abdechakour Elkihel, Jing Wu, Ling Peng, Carlo Vascotto

{"title":"Quantitative analysis of oligonucleotide delivery into isolated mitochondria: a proof-of-concept method.","authors":"Joshua McHale, Veronica Bazzani, Abdechakour Elkihel, Jing Wu, Ling Peng, Carlo Vascotto","doi":"10.1080/07366205.2026.2635461","DOIUrl":"10.1080/07366205.2026.2635461","url":null,"abstract":"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.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"1-11"},"PeriodicalIF":2.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147509486","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}

引用次数: 0

Comparative performance of high-throughput qPCR, qPCR, and digital PCR for antibiotic resistance gene monitoring in tropical water systems. 高通量qPCR、qPCR和数字PCR在热带水系统抗生素耐药基因监测中的比较性能

IF 2.5 4区工程技术

BioTechniques Pub Date : 2026-01-01 Epub Date: 2026-04-22 DOI: 10.1080/07366205.2026.2649861

Thitima Srathongneam, Phongsawat Paisantham, Andrew C Singer, Rojana Sukchawalit, Skorn Mongkolsuk, Kwanrawee Sirikanchana

{"title":"Comparative performance of high-throughput qPCR, qPCR, and digital PCR for antibiotic resistance gene monitoring in tropical water systems.","authors":"Thitima Srathongneam, Phongsawat Paisantham, Andrew C Singer, Rojana Sukchawalit, Skorn Mongkolsuk, Kwanrawee Sirikanchana","doi":"10.1080/07366205.2026.2649861","DOIUrl":"10.1080/07366205.2026.2649861","url":null,"abstract":"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.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"158-169"},"PeriodicalIF":2.5,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147760898","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}

引用次数: 0

Evaluation and verification of cfDNA extraction methods. cfDNA提取方法的评价与验证。

IF 2.5 4区工程技术

BioTechniques Pub Date : 2026-01-01 Epub Date: 2026-05-04 DOI: 10.1080/07366205.2026.2665280

Cecilie Mondrup Jacobsen, Luisa Matos do Canto, Estrid Høgdall, Rikke Fredslund Andersen

引用次数: 0

High-resolution molecular typing of vancomycin-resistant Enterococcus faecium from Romania and Bavaria: combining enhanced DNA microarray and next generation sequencing. 罗马尼亚和巴伐利亚耐万古霉素屎肠球菌的高分辨率分子分型：结合增强DNA微阵列和下一代测序。

IF 2.6 4区工程技术

BioTechniques Pub Date : 2026-01-01 Epub Date: 2026-02-02 DOI: 10.1080/07366205.2026.2621054

Ibukun Elizabeth Osadare, Abdinasir Abdilahi, Elke Müller, Mara Lohde, Celia Diezel, Maximilian Collatz, Sascha Braun, Bärbel Kieninger, Anja Eichner, Wulf Schneider-Brachert, Thomas Wellhöfer, Katrin Frankenfeld, Olivia Dorneanu, Stefan Monecke, Ralf Ehricht

{"title":"High-resolution molecular typing of vancomycin-resistant Enterococcus faecium from Romania and Bavaria: combining enhanced DNA microarray and next generation sequencing.","authors":"Ibukun Elizabeth Osadare, Abdinasir Abdilahi, Elke Müller, Mara Lohde, Celia Diezel, Maximilian Collatz, Sascha Braun, Bärbel Kieninger, Anja Eichner, Wulf Schneider-Brachert, Thomas Wellhöfer, Katrin Frankenfeld, Olivia Dorneanu, Stefan Monecke, Ralf Ehricht","doi":"10.1080/07366205.2026.2621054","DOIUrl":"10.1080/07366205.2026.2621054","url":null,"abstract":"Antimicrobial resistance poses a significant challenge for infection control, requiring the development of accurate and high-throughput diagnostic techniques. We expanded and optimized an existing DNA microarray platform for the molecular characterization of vancomycin-resistant Enterococcus (VRE) by incorporating resistance, virulence, species-specific, and typing markers. The enhanced microarray allows for the simultaneous analysis of up to 96 strains, providing detailed genetic profiles of clinical isolates. VRE strains from Romania and Bavaria, Germany, were analyzed, and the results were compared to those obtained using traditional typing methods, such as multilocus sequence typing (MLST). Next-generation sequencing (NGS) was used in parallel to validate the microarray findings and explore genomic relationships. The microarray revealed considerable genetic diversity and potential epidemiological linkages among isolates. A novel hexadecimal-based nomenclature system was introduced for standardized and scalable strain classification. Comparative analysis demonstrated that the array profiles provided greater discriminatory power and practical resolution than MLST. Receiver operating characteristic (ROC) curve analysis of 187 target genes in 220 isolates gave diagnostic sensitivity and specificity of 100%. This integrated approach offers a cost-effective, rapid, and adaptable global VRE surveillance and infection control tool. It provides a practical alternative to conventional typing systems and facilitates early detection of outbreaks and emerging clones.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"1-15"},"PeriodicalIF":2.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146103718","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}

引用次数: 0

Strand-specific quantification of L1 ORF0 and related transcripts by multiplex reverse transcription with tagged primers. 用带标记引物多重反转录对L1 ORF0及相关转录物进行链特异性定量。

IF 2.6 4区工程技术

BioTechniques Pub Date : 2026-01-01 Epub Date: 2026-06-08 DOI: 10.1080/07366205.2026.2678311

Kazunari Mizuno, Yutaka Nakachi, Emi Kiyota, Wataru Ukai, Takao Ishii, Eri Hashimoto, Miki Bundo, Kazuya Iwamoto

{"title":"Strand-specific quantification of L1 ORF0 and related transcripts by multiplex reverse transcription with tagged primers.","authors":"Kazunari Mizuno, Yutaka Nakachi, Emi Kiyota, Wataru Ukai, Takao Ishii, Eri Hashimoto, Miki Bundo, Kazuya Iwamoto","doi":"10.1080/07366205.2026.2678311","DOIUrl":"10.1080/07366205.2026.2678311","url":null,"abstract":"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.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"253-262"},"PeriodicalIF":2.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148203929","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}

引用次数: 0