BioTechniquesPub Date : 2024-11-01Epub Date: 2024-12-25DOI: 10.1080/07366205.2024.2445454
Nicole Rodriguez Ortiz, Niharika Sharma, Tian-Xiong Zheng, James J Campanella
{"title":"Characterization of nuclear microsatellites in <i>Marchantia polymorpha</i> (liverwort) with additional trans-specific analyses.","authors":"Nicole Rodriguez Ortiz, Niharika Sharma, Tian-Xiong Zheng, James J Campanella","doi":"10.1080/07366205.2024.2445454","DOIUrl":"10.1080/07366205.2024.2445454","url":null,"abstract":"<p><p>Microsatellites are present in mitochondria, chloroplast, and nuclear DNA, but nuclear microsatellites are more useful genetic tools than those in plastids or mitochondria. Plastid and mitochondrial microsatellites have been identified in the model plant <i>Marchantia polymorpha</i> (liverwort), but no laboratory has published information on nuclear microsatellite loci. The aim of this study was to detect novel nuclear markers in the most commonly employed liverwort species, design PCR primers that would allow amplification, and characterize the subsequently generated loci. We detected 18 polymorphic nuclear loci in <i>M. polymorpha</i>, amplifiable by PCR across all chromosomes. Additionally, trans-specific amplification of the eighteen loci was characterized in the closely related taxa <i>Marchantia emarginata</i> and <i>Marchantia paleacea</i>. All loci were present in <i>M. paleacea</i>, whereas 17 of 18 primer pairs were amplified in <i>M. emarginata</i>.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"527-534"},"PeriodicalIF":2.2,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142891802","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}
BioTechniquesPub Date : 2024-11-01Epub Date: 2024-12-16DOI: 10.1080/07366205.2024.2441637
Gengxuan Shi, Yunjiang Feng, Kathryn F Tonissen
{"title":"Development of a human tyrosinase activity inhibition assay using human melanoma cell lysate.","authors":"Gengxuan Shi, Yunjiang Feng, Kathryn F Tonissen","doi":"10.1080/07366205.2024.2441637","DOIUrl":"10.1080/07366205.2024.2441637","url":null,"abstract":"<p><p>Tyrosinase (TYR) inhibitors are required to treat skin hyperpigmentation. Currently live cell-based TYR assays and mushroom TYR <i>in vitro</i> assays are the common methods used to screen for TYR inhibitors. However, these methods are either time consuming and expensive or are not human TYR (<i>hs</i>TYR) specific. Here, we describe a simple <i>hs</i>TYR assay using cell lysate prepared from pigmented human melanoma cell lines that takes less than 3 hours to complete after collecting cell pellets. We confirmed the assay is species specific by using a known <i>hs</i>TYR inhibitor, kojic acid, as a positive control, while arbutin, which inhibits mushroom TYR, but not <i>hs</i>TYR, was not effective. This assay is a simple method to confirm <i>hs</i>TYR inhibition before conducting follow-up studies in live biological models.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"547-551"},"PeriodicalIF":2.2,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142827092","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}
BioTechniquesPub Date : 2024-11-01Epub Date: 2025-01-06DOI: 10.1080/07366205.2024.2445467
Annie Coulson
{"title":"Who wants to live forever? Models shaping the future of aging research.","authors":"Annie Coulson","doi":"10.1080/07366205.2024.2445467","DOIUrl":"10.1080/07366205.2024.2445467","url":null,"abstract":"","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"523-526"},"PeriodicalIF":2.2,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142930578","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}
BioTechniquesPub Date : 2024-10-01Epub Date: 2024-12-15DOI: 10.1080/07366205.2024.2432805
Silke Fähnrich, Anne Wedemann, Laura Steenpass, Wilhelm Gerhard Dirks
{"title":"Optimized for routine: highly sensitive fluorescent Telomeric Repeat Amplification Protocol (f-TRAP).","authors":"Silke Fähnrich, Anne Wedemann, Laura Steenpass, Wilhelm Gerhard Dirks","doi":"10.1080/07366205.2024.2432805","DOIUrl":"10.1080/07366205.2024.2432805","url":null,"abstract":"<p><p>The strict suppression of telomerase activity (TA) in terminally differentiated human cells causes a shortening of the chromosome ends after each cell division. This tumor suppression surveillance mechanism is associated with a limited number of cell divisions known as Hayflick limit. Here we present an optimized protocol for measuring TA that combines a fluorescently labeled bait primer and polymerase chain reaction (PCR) amplification with analytical capillary electrophoresis (CE) to achieve a detection limit of one telomerase-positive cell per ten thousand negative cells. In research laboratories today, a broad panel of TRAP assay protocols enables the assessment of the immortality of newly generated cell lines or the unambiguous evaluation of the reprogramming of induced pluripotent stem cells (iPSCs). The present f-TRAP protocol, optimized for routine use, enables fast ad hoc application for single measurements up to a high throughput of mass samples using a triplicate approach of different lysate concentrations. Final CE analysis facilitates standardized data processing and storage for documentation of results and could make f-TRAP a useful assay in research and clinical oncology.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"517-522"},"PeriodicalIF":2.2,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142827093","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}
BioTechniquesPub Date : 2024-10-01Epub Date: 2024-12-04DOI: 10.1080/07366205.2024.2429308
Zhi-Xiang Lu, Anis H Khimani, Yanhong Tong
{"title":"Importance of a reference ladder in analytical testing of CRISPR HDR donor ssDNA templates.","authors":"Zhi-Xiang Lu, Anis H Khimani, Yanhong Tong","doi":"10.1080/07366205.2024.2429308","DOIUrl":"10.1080/07366205.2024.2429308","url":null,"abstract":"","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"479-484"},"PeriodicalIF":2.2,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142779185","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}
BioTechniquesPub Date : 2024-10-01Epub Date: 2024-12-02DOI: 10.1080/07366205.2024.2427544
Anthony J Politza, Tianyi Liu, Aneesh Kshirsagar, Ming Dong, Md Ahasan Ahamed, Weihua Guan
{"title":"Development and validation of a portable device for lab-free versatile nucleic acid extraction.","authors":"Anthony J Politza, Tianyi Liu, Aneesh Kshirsagar, Ming Dong, Md Ahasan Ahamed, Weihua Guan","doi":"10.1080/07366205.2024.2427544","DOIUrl":"10.1080/07366205.2024.2427544","url":null,"abstract":"<p><p>Nucleic acid testing (NAT) has revolutionized diagnostics by providing precise, rapid, and scalable detection methods for diverse biological samples. These recent advancements satisfy the increasing demand for on-site diagnostics, yet sample preparation remains a significant bottleneck for achieving highly sensitive diagnostic assays. There is an unmet need for compatible, efficient, and lab-free sample preparation for point-of-care NAT. To address this, we developed a portable, lab-free, and battery-powered device for extracting nucleic acids. We explored using low centrifugal forces with existing commercial chemistry, demonstrating excellent performance. We designed and tested a battery-powered device to enable lab-free extractions, and verified reagents stored out to 6 months, suggesting exceptional deployment capabilities. We evaluated our device, comparing our results against those from a benchtop centrifuge across three types of samples: HIV RNA in buffer, HIV RNA in plasma, and SARS-CoV-2 RNA in saliva. The portable device demonstrated excellent agreement with the benchtop centrifuge, indicating high reliability. By providing an effective on-site sample preparation solution, the widespread adoption of low centrifugal extractions will improve the sensitivity and reliability of NAT and will positively impact other point-of-care technologies such as next generation sequencing (NGS), biomarker detection, and environmental monitoring.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"505-515"},"PeriodicalIF":2.2,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142765762","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":"A single-cell 3D dynamic volume control system for chondrocytes.","authors":"Qiang Zhang, Yiyao Wang, Yanjun Zhang, Xiaochun Wei, Weiyi Chen, Quanyou Zhang","doi":"10.1080/07366205.2024.2412414","DOIUrl":"10.1080/07366205.2024.2412414","url":null,"abstract":"<p><p>In articular cartilage, zone-specific cellular morphology is a typical characteristic of cartilage tissue, which is related with chondrocyte function, inflammation and osteoarthritis (OA). Chondrocyte hypertrophic phenotype is a criticle physiological process which indicates a hallmark of chondrocyte terminal differentiation and bone formation. Thus, developing a <i>in vitro</i> cell culture system for dynamic regulation of single chondrocyte volume at a three-dimensional (3D) level is particularly necessary for understanding how physical cues of matrix microenvironment regulate chondrocyte fate and the degeneration of articular cartilage. Here, based on the soft lithography techniques, we have constructed well-defined single-cell 3D dynamic volume control system to recapitulate the physiological matrix microenvironment of single chondrocyte niche. The results of finite element analysis indicated that the stress and strain distribution in the cell culture region is homogeneous during the stretching process. Additionally, 3D dynamic volume expansion and compression of single cells in physiological or hyperphysiological can be realized in this cell culture system. Our device for single-cell 3D dynamic culture provides a microphysiological culture system for chondrocytes to explore the mechanisms of cartilage hypertrophy, as well as develops a new paradigm for functional cartilage tissue engineering and regenerative medicine.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"495-504"},"PeriodicalIF":2.2,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142457178","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}
BioTechniquesPub Date : 2024-10-01Epub Date: 2024-11-28DOI: 10.1080/07366205.2024.2418748
Louis J Gautier
{"title":"CRISPR in 3D: Innovations in Disease Modelling and Personalized Medicine.","authors":"Louis J Gautier","doi":"10.1080/07366205.2024.2418748","DOIUrl":"10.1080/07366205.2024.2418748","url":null,"abstract":"<p><p>The CRISPR-Cas system of genetic engineering has had a significant impact on science and society since its advent in 2013. CRISPR integration with 3D culture systems such as organ-on-a-chips, as well as fast emerging commercial technologies, has encouraged translation of more complex pathophysiological modelling and personalized medicine.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"473-478"},"PeriodicalIF":2.2,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142738183","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}
BioTechniquesPub Date : 2024-09-13DOI: 10.1080/07366205.2024.2397284
James D Feller,Leah Colton
{"title":"Comparison of commercially available DNA and RNA extraction kits for wildlife feces collected from the environment.","authors":"James D Feller,Leah Colton","doi":"10.1080/07366205.2024.2397284","DOIUrl":"https://doi.org/10.1080/07366205.2024.2397284","url":null,"abstract":"Wildlife fecal samples were collected across two Air Force installations to evaluate the effectiveness of commercially available DNA and RNA extraction kits. Four DNA kits, two DNA/RNA kits and one RNA only kit were used. Sample extracts were evaluated on nucleic acid concentration, TapeStation DNA or RNA Integrity Number values and presence of PCR inhibitors. For the DNA kits, PFP produced higher concentrations compared with PLM and RPM, while MWFM gave higher DNA Integrity Number values when compared with RPM. No PCR inhibition was detected. For the RNA kits, RPM gave higher concentrations compared with MWTV and no differences were seen in RNA Integrity Number values. PCR inhibition was observed in all RNA samples, with MWTV exhibiting higher inhibition compared with RPM.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"1 1","pages":"1-10"},"PeriodicalIF":2.7,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142248665","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}
BioTechniquesPub Date : 2024-09-13DOI: 10.1080/07366205.2024.2397288
Sonia Mukherjee,Pierre Leblanc,Mark C Poznansky,Ann E Sluder
{"title":"A HABA dye-based colorimetric assay to detect unoccupied biotin binding sites in an avidin-containing fusion protein.","authors":"Sonia Mukherjee,Pierre Leblanc,Mark C Poznansky,Ann E Sluder","doi":"10.1080/07366205.2024.2397288","DOIUrl":"https://doi.org/10.1080/07366205.2024.2397288","url":null,"abstract":"Avidin-biotin binding, the most robust non-covalent protein-ligand interaction occurring in nature, has wide-ranging applications in biotechnology. A frequent challenge in these applications is accurately determining the number of unoccupied biotin binding sites in avidin-containing fusion proteins. We delineate a novel assay protocol in miniaturized format to quantify available biotin binding sites based on the affinity of the anionic dye 4'-hydroxyazobenzene-2-carboxylic acid for biotin binding sites within avidin. We apply this assay as a quality control assay to evaluate the number of available biotin binding sites in different fusion protein production batches. This method offers a streamlined alternative to fluorescence-based assays commonly employed to assess biotin binding, is less time-consuming than other methods and is applicable to diverse fusion proteins.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"22 1","pages":"1-10"},"PeriodicalIF":2.7,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142248664","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}