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pJoseph2: a family of plasmids as positive controls for bacterial protein expression, transfections, and western blots. pJoseph2：质粒系列，可作为细菌蛋白质表达、转染和 Western 印迹的阳性对照。
IF 2.2 4区 工程技术
BioTechniques Pub Date : 2024-01-01 Epub Date: 2024-05-12 DOI: 10.1080/07366205.2024.2343609
Ebru Robinson, Elizabeth Barajas Alonso, Jennifer A Waters, Cayleen Bileckyj, Carrie D House, Christopher A Johnston, Richard M Cripps
{"title":"pJoseph2: a family of plasmids as positive controls for bacterial protein expression, transfections, and western blots.","authors":"Ebru Robinson, Elizabeth Barajas Alonso, Jennifer A Waters, Cayleen Bileckyj, Carrie D House, Christopher A Johnston, Richard M Cripps","doi":"10.1080/07366205.2024.2343609","DOIUrl":"10.1080/07366205.2024.2343609","url":null,"abstract":"<p><p>Epitope tagging represents a powerful strategy for expedited identification, isolation, and characterization of proteins in molecular biological studies, including protein-protein interactions. We aimed to improve the reproducibility of epitope-tagged protein expression and detection by developing a range of plasmids as positive controls. The pJoseph2 family of expression plasmids functions in diverse cellular environments and cell types to enable the evaluation of transfection efficiency and antibody staining for epitope detection. The expressed green fluorescent proteins harbor five unique epitope tags, and their efficient expression in <i>Escherichia coli</i>, <i>Drosophila</i> Schneider's line 2 cells, and human SKOV3 and HEK293T cells was demonstrated by fluorescence microscopy and western blotting. The pJoseph2 plasmids provide versatile and valuable positive controls for numerous experimental applications.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"76 7","pages":"299-309"},"PeriodicalIF":2.2,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11796145/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142054860","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Modular probe-based colorimetric miRNA detection via polymerase/endonuclease assisted chain displacement. 通过聚合酶/内切酶辅助链置换进行基于探针的模块化比色 miRNA 检测。
IF 2.2 4区 工程技术
BioTechniques Pub Date : 2024-01-01 Epub Date: 2024-07-23 DOI: 10.1080/07366205.2024.2368394
Xialing Xu, Ping Zhang, Siyu Tao
{"title":"Modular probe-based colorimetric miRNA detection <i>via</i> polymerase/endonuclease assisted chain displacement.","authors":"Xialing Xu, Ping Zhang, Siyu Tao","doi":"10.1080/07366205.2024.2368394","DOIUrl":"10.1080/07366205.2024.2368394","url":null,"abstract":"<p><p>Methods for sequence-specific microRNA (miRNA) analysis are crucial for miRNA research and guiding nursing strategies. We have devised a colorimetric technique for detecting miRNA using a dumbbell probe-based polymerase/endonuclease assisted chain displacement, along with silver ions (Ag<sup>+</sup>) aptamer assisted color reaction. The suggested approach enables precise measurement of miRNA-21 within the concentration range of 100 fM-5 nM, with a low detection limit of 45.32 fM. Additionally, it exhibits exceptional capability in distinguishing variations at the level of individual nucleotides. Furthermore, the detection technique may be utilized to precisely measure the amount of miRNA-21 in serum samples, demonstrating a high level of concordance with the findings obtained from a commercially available miRNA detection kit.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"371-379"},"PeriodicalIF":2.2,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141747399","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
Isothermal amplification of long DNA fragments at low temperature by improved strand displacement amplification. 通过改进的链置换扩增技术在低温下等温扩增长 DNA 片段。
IF 2.2 4区 工程技术
BioTechniques Pub Date : 2024-01-01 Epub Date: 2024-03-28 DOI: 10.2144/btn-2024-0012
Yinhua Zhang, Ashley N Luck, Nathan A Tanner
{"title":"Isothermal amplification of long DNA fragments at low temperature by improved strand displacement amplification.","authors":"Yinhua Zhang, Ashley N Luck, Nathan A Tanner","doi":"10.2144/btn-2024-0012","DOIUrl":"10.2144/btn-2024-0012","url":null,"abstract":"<p><p>Strand displacement amplification (SDA) is an isothermal amplification technique wherein amplification of a nucleic acid is initiated by nicking enzyme activity at sites flanking the target. Diagnostic SDA is very fast but requires precise optimization and is limited to very short amplicons. Here we report an enhanced approach by addition of single-stranded DNA binding protein, crowding agents and dUTP to enable amplification of kilobase-length products at low temperatures. Additionally, we pair this improved SDA with a novel carryover contamination prevention, eliminating amplifiable DNA at the end of the reaction to reduce contamination risk. Taken together these developments increase the utility and versatility of SDA, broadening the reach of this powerful but uncommonly used method.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"255-262"},"PeriodicalIF":2.2,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140304764","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
Decoding disease: Scott Patterson's perspectives on the power of biomarkers in drug development. 解码疾病:斯科特·帕特森对药物开发中生物标志物力量的看法。
IF 2.7 4区 工程技术
BioTechniques Pub Date : 2024-01-01 Epub Date: 2023-11-23 DOI: 10.2144/btn-2023-0108
Scott D Patterson
{"title":"Decoding disease: Scott Patterson's perspectives on the power of biomarkers in drug development.","authors":"Scott D Patterson","doi":"10.2144/btn-2023-0108","DOIUrl":"10.2144/btn-2023-0108","url":null,"abstract":"<p><p>Scott Patterson (Gilead Sciences Inc., CA, USA) speaks to Ashling Cannon, Journal Development Editor at <i>BioTechniques</i>, about his career. Patterson is a biochemist and proteomics and biomarker/translational expert with over 30 years of industry experience following 13 years in an academic setting. Patterson earned his BSc and PhD in Physiology and Pharmacology from the University of Queensland (Australia) while working full time in the Department of Physiology and Pharmacology, rising to a Senior Research Officer. Throughout his career, Patterson has been actively involved in advancing technologies, how they can be applied to address biological questions and the interplay of bioinformatics and large datasets leveraging biomarkers and diagnostics. He has held pivotal roles at renowned institutions and companies such as Cold Spring Harbor Laboratory (NY, USA), Amgen, Inc. (CA, USA), Celera Genomics Group (MD, USA) and Gilead Sciences, Inc. Notably, he served as a Staff Investigator at Cold Spring Harbor Laboratory and was honored with the Long Island Biological Association New Investigator award in addition to being the 2002 Barnett Lecturer at Northeastern University (MA, USA). In early 2015 Patterson joined Gilead Sciences, Inc., bringing his extensive expertise to lead biomarker discovery and development as well as <i>in vitro</i> diagnostics initiatives across all therapeutic domains.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"9-13"},"PeriodicalIF":2.7,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138294493","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
Three-dimensional visualization and analysis of dendritic spines in human brain tissue. 人脑组织树突棘的三维可视化与分析。
IF 2.7 4区 工程技术
BioTechniques Pub Date : 2024-01-01 Epub Date: 2023-11-23 DOI: 10.2144/btn-2023-0078
Haitao Sun, Hei Ming Lai, Wutian Wu
{"title":"Three-dimensional visualization and analysis of dendritic spines in human brain tissue.","authors":"Haitao Sun, Hei Ming Lai, Wutian Wu","doi":"10.2144/btn-2023-0078","DOIUrl":"10.2144/btn-2023-0078","url":null,"abstract":"<p><p>We developed a simple yet powerful technique to visualize neuronal morphology in human brain tissues. By ballistically shooting DiI (1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate)-coated tungsten particles to randomly label neurons, then clearing tissues with OPTIClear, we demonstrated the tracing of branched dendritic trees and spines in three dimensions. High-resolution imaging revealed dendrites up to 300 μm long and spine necks down to 200 nm across. Quantitative analyses of 1304 dendritic spines showed no decrease in spine density with imaging depth, indicating excellent clearing and tracing. Segmentation and modeling of dendritic spines enabled morphological characterization. This technique enables assumption-free, high-resolution and cost-efficient visualization of neuronal morphology in human tissues. Combined with immunohistochemistry and electron microscopy, it could provide new perspectives for studying human neuroanatomy and pathology.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"37-42"},"PeriodicalIF":2.7,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138294494","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
Reference genes for gene expression profiling in mouse models of Listeria monocytogenes infection. 用于李斯特菌感染小鼠模型基因表达谱分析的参考基因。
IF 2.7 4区 工程技术
BioTechniques Pub Date : 2023-12-19 DOI: 10.2144/btn-2023-0063
Lethicia Souza Tavares, Roberta Lane Oliveira-Silva, Marcelo Tigre Moura, Jéssica Barboza da Silva, Ana Maria Benko-Iseppon, José Vitor Lima-Filho
{"title":"Reference genes for gene expression profiling in mouse models of Listeria monocytogenes infection.","authors":"Lethicia Souza Tavares, Roberta Lane Oliveira-Silva, Marcelo Tigre Moura, Jéssica Barboza da Silva, Ana Maria Benko-Iseppon, José Vitor Lima-Filho","doi":"10.2144/btn-2023-0063","DOIUrl":"https://doi.org/10.2144/btn-2023-0063","url":null,"abstract":"RT-qPCR dissects transcription-based processes but requires reference genes (RGs) for data normalization. This study prospected RGs for mouse macrophages (pMØ) and spleen infected with <i>Listeria monocytogenes</i>. The pMØ were infected <i>in vitro</i> with <i>L. monocytogenes</i> or vehicle for 4 h. Mice were injected with <i>L. monocytogenes</i> (or vehicle) and euthanized 24 h post-injection. The RGs came from a multispecies primer set, from the literature or designed here. The RG ranking relied on GeNorm, NormFinder, BestKeeper, Delta-CT and RefFinder. <i>B2m</i>-<i>H3f3a</i>-<i>Ppia</i> were the most stable RGs for pMØ, albeit RG indexes fine-tuned estimations of cytokine relative expression. <i>Actβ-Ubc</i>-<i>Ppia</i> were the best RGs for spleen but modestly impacted the cytokine relative expression. Hence, mouse models of <i>L. monocytogenes</i> require context-specific RGs for RT-qPCR, thus reinforcing its paramount contribution to accurate gene expression profiling.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"2 1","pages":""},"PeriodicalIF":2.7,"publicationDate":"2023-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138745332","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
Cradle cultures: growing stem cell-derived developmental cell models in vitro. 摇篮培养:体外培养干细胞衍生的发育细胞模型。
IF 2.7 4区 工程技术
BioTechniques Pub Date : 2023-12-01 Epub Date: 2023-11-16 DOI: 10.2144/btn-2023-0100
Beatrice Bowlby
{"title":"Cradle cultures: growing stem cell-derived developmental cell models <i>in vitro</i>.","authors":"Beatrice Bowlby","doi":"10.2144/btn-2023-0100","DOIUrl":"10.2144/btn-2023-0100","url":null,"abstract":"<p><p>How are three stem cell-derived developmental cell models furthering our understanding of post-implantation human embryo development? And why have recent advancements in these human embryo-like models spurred ethical discussion and the need to refine our definition of 'embryo'? [Formula: see text].</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"227-230"},"PeriodicalIF":2.7,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134648420","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 simplified and rapid in situ hybridization protocol for planarians. 一种用于涡虫的简化快速原位杂交方案。
IF 2.7 4区 工程技术
BioTechniques Pub Date : 2023-12-01 Epub Date: 2023-10-18 DOI: 10.2144/btn-2023-0074
Andrew J Gaetano, Ryan S King
{"title":"A simplified and rapid <i>in situ</i> hybridization protocol for planarians.","authors":"Andrew J Gaetano, Ryan S King","doi":"10.2144/btn-2023-0074","DOIUrl":"10.2144/btn-2023-0074","url":null,"abstract":"<p><p>Whole-mount <i>in situ</i> hybridization is a critical technique for analyzing gene expression in planarians. While robust <i>in situ</i> protocols have been developed, these protocols are laborious, making them challenging to incorporate in an academic setting, reducing throughput and increasing time to results. Here, the authors systematically tested modifications to all phases of the protocol with the goal of eliminating steps and reducing time without impacting quality. This modified protocol allows for whole-mount colorimetric <i>in situ</i> hybridization and multicolor fluorescence <i>in situ</i> hybridization to be completed in two days with a significant reduction in steps and hands-on processing time.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"231-239"},"PeriodicalIF":2.7,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41232086","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
An easy method for quantification of anaerobic and microaerobic gene expression with fluorescent reporter proteins. 一种用荧光报告蛋白定量厌氧和微需氧基因表达的简单方法。
IF 2.7 4区 工程技术
BioTechniques Pub Date : 2023-12-01 Epub Date: 2023-10-26 DOI: 10.2144/btn-2023-0064
Lucas Pedraz, Eduard Torrents
{"title":"An easy method for quantification of anaerobic and microaerobic gene expression with fluorescent reporter proteins.","authors":"Lucas Pedraz, Eduard Torrents","doi":"10.2144/btn-2023-0064","DOIUrl":"10.2144/btn-2023-0064","url":null,"abstract":"<p><p>Fluorescent proteins, such as green fluorescent proteins, are invaluable tools for detecting and quantifying gene expression in high-throughput reporter gene assays. However, they introduce significant inaccuracies in studies involving microaerobiosis or anaerobiosis, as oxygen is required for the maturation of these proteins' chromophores. In this study, the authors highlight the errors incurred by using fluorescent proteins under limited oxygenation by comparing standard fluorescence-based reporter gene assays to quantitative real-time PCR data in the study of a complex oxygen-regulated gene network. Furthermore, a solution to perform quantification of anaerobic and microaerobic gene expression with fluorescent reporter proteins using a microplate reader with an oxygen control system and applying pulses of full oxygenation before fluorescence measurements is provided.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"250-255"},"PeriodicalIF":2.7,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50160553","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
Simple, fast and inexpensive hot sodium hydroxide and tris DNA extraction method for genotyping tomato and melon seeds. 简单、快速、廉价的热氢氧化钠和三链DNA提取方法用于番茄和瓜子的基因分型。
IF 2.7 4区 工程技术
BioTechniques Pub Date : 2023-12-01 Epub Date: 2023-11-02 DOI: 10.2144/btn-2023-0054
Yolanda García-Abolafio, Francisco Villanueva, María Urrutia
{"title":"Simple, fast and inexpensive hot sodium hydroxide and tris DNA extraction method for genotyping tomato and melon seeds.","authors":"Yolanda García-Abolafio, Francisco Villanueva, María Urrutia","doi":"10.2144/btn-2023-0054","DOIUrl":"10.2144/btn-2023-0054","url":null,"abstract":"<p><p>Seed commerce is a highly profitable global market. Most commercialized seeds are hybrid seeds originating from a controlled cross between two selected parental lines. The market value of hybrid seeds depends on their hybrid genetic purity. DNA molecular markers are a reliable and widespread tool to genotype plant materials; however, DNA extraction from seeds is challenging, often laborious and expensive. With the ultimate goal of creating a tomato and melon hybrid seeds purity test, various challenges arise. To overcome these problems and with the purpose of crude DNA extraction, a simple, fast, inexpensive and easily scalable adaptation of the hot sodium hydroxide and tris method coupled to a competitive allele-specific PCR genotyping method is proposed.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"245-249"},"PeriodicalIF":2.7,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"71420365","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
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