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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
Considerations for colorblind individuals on selecting colorimetric or fluorescent dye assay outcomes. 色盲个体选择比色或荧光染料测定结果的注意事项。
IF 2.7 4区 工程技术
BioTechniques Pub Date : 2023-12-01 Epub Date: 2023-11-09 DOI: 10.2144/btn-2023-0041
Kirstyn Loyva, Erik Hofmeister, Fiona Georgousi, Constance Roderick, Rebecca Cole
{"title":"Considerations for colorblind individuals on selecting colorimetric or fluorescent dye assay outcomes.","authors":"Kirstyn Loyva, Erik Hofmeister, Fiona Georgousi, Constance Roderick, Rebecca Cole","doi":"10.2144/btn-2023-0041","DOIUrl":"10.2144/btn-2023-0041","url":null,"abstract":"<p><p>A disadvantage of colorimetric detection in nucleic acid amplification assays is the possibility that a colorblind individual may interpret colors differently than observers with full-color vision. Using an isothermal amplification assay, the ability of colorblind individuals to distinguish between positive and negative results for four dyes was tested. Five individuals with self-reported colorblindness and four with full-color vision reported their observations of the color of the solution. Although colorblind individuals may accurately interpret assay results, they were often not accurate in reporting the color. Hydroxynaphthol blue was the most problematic dye, and both phenol red and SYBR™ green were less troublesome. Consideration for colorblind individuals is warranted when developing an assay and training staff in its performance.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"240-244"},"PeriodicalIF":2.7,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"71520388","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 simple, sensitive and colorimetric assay for Pseudomonas aeruginosa infection analysis. 一种用于铜绿假单胞菌感染分析的简单、灵敏和比色测定法。
IF 2.7 4区 工程技术
BioTechniques Pub Date : 2023-11-01 Epub Date: 2023-10-26 DOI: 10.2144/btn-2023-0030
Jie Liu, Dan Lu, Junyuan Wang
{"title":"A simple, sensitive and colorimetric assay for <i>Pseudomonas aeruginosa</i> infection analysis.","authors":"Jie Liu, Dan Lu, Junyuan Wang","doi":"10.2144/btn-2023-0030","DOIUrl":"10.2144/btn-2023-0030","url":null,"abstract":"<p><p>Skin and soft tissue infections caused by <i>Pseudomonas aeruginosa</i> are common acquired diseases in postpartum care. Many methods have been developed in recent years for detecting <i>P. aeruginosa</i>, but they are criticized for the drawbacks of labor-intensiveness, complicated operation and high cost. Here, a simple, sensitive and colorimetric assay for <i>P. aeruginosa</i> detection is described. The approach displays a green color for positive samples and colorless for target-free samples. The approach exhibits a wide detection range and a low limit of detection of 45 CFU/ml. Thus, the developed ligation-initiated multiple-signal amplification method may be used for on-site testing of pathogenic bacteria and assist in the early diagnosis of postpartum care skin infections.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"210-217"},"PeriodicalIF":2.7,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50160552","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 the mechanisms of embryo development with spatial biology. 用空间生物学解读胚胎发育机制。
IF 2.7 4区 工程技术
BioTechniques Pub Date : 2023-11-01 Epub Date: 2023-10-19 DOI: 10.2144/btn-2023-0093
Asisha Al-Janabi
{"title":"Decoding the mechanisms of embryo development with spatial biology.","authors":"Asisha Al-Janabi","doi":"10.2144/btn-2023-0093","DOIUrl":"10.2144/btn-2023-0093","url":null,"abstract":"<p><p>[Formula: see text] Spatial genomics first emerged in the 1990s to map biological function data onto multidimensional images of embryo specimens and has continued to be instrumental in studying embryogenesis and understanding early human development in the decades since.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"179-182"},"PeriodicalIF":2.7,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49673945","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
Generation of UL128-shRNA transduced fibroblasts for the release of cell-free virus from clinical human cytomegalovirus isolates. 产生UL128 shRNA转导的成纤维细胞，用于从临床人类巨细胞病毒分离株中释放无细胞病毒。
IF 2.7 4区 工程技术
BioTechniques Pub Date : 2023-11-01 Epub Date: 2023-10-17 DOI: 10.2144/btn-2023-0046
Nina Weiler, Kerstin Laib Sampaio, Myriam Scherer, Christian Sinzger
{"title":"Generation of <i>UL128</i>-shRNA transduced fibroblasts for the release of cell-free virus from clinical human cytomegalovirus isolates.","authors":"Nina Weiler, Kerstin Laib Sampaio, Myriam Scherer, Christian Sinzger","doi":"10.2144/btn-2023-0046","DOIUrl":"10.2144/btn-2023-0046","url":null,"abstract":"<p><p>Working with recent isolates of human cytomegalovirus (HCMV) is complicated by their strictly cell-associated growth with lack of infectivity in the supernatant. Adaptation to cell-free growth is associated with disruption of the viral <i>UL128</i> gene locus. The authors transduced fibroblasts with a lentiviral vector encoding <i>UL128</i>-specific-shRNA to allow the release of cell-free infectivity without genetic alteration. Transduced cells were cocultured with fibroblasts containing cell-associated isolates, and knockdown of the UL128 protein was validated by immunoblotting. Cell-free infectivity increased 1000-fold in isolate cocultures with <i>UL128</i>-shRNA compared with controls, and virions could be purified by density gradients. Transduced fibroblasts also allowed direct isolation of HCMV from a clinical specimen and cell-free transfer to other cell types. In conclusion, <i>UL128</i>-shRNA-transduced fibroblasts allow applications previously unsuitable for recent isolates.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"183-194"},"PeriodicalIF":2.7,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41232087","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 simple and rapid method for isolating high-quality RNA from kenaf with high polysaccharide and polyphenol contents. 一种从红麻中分离高质量多糖和多酚RNA的简单快速方法。
IF 2.7 4区 工程技术
BioTechniques Pub Date : 2023-11-01 Epub Date: 2023-10-26 DOI: 10.2144/btn-2023-0077
Xiaofang Liao, Yanhong Zhao, Hongwei Li, Wenhuan Hou, Xingfu Tang, Ruiyang Zhou
{"title":"A simple and rapid method for isolating high-quality RNA from kenaf with high polysaccharide and polyphenol contents.","authors":"Xiaofang Liao, Yanhong Zhao, Hongwei Li, Wenhuan Hou, Xingfu Tang, Ruiyang Zhou","doi":"10.2144/btn-2023-0077","DOIUrl":"10.2144/btn-2023-0077","url":null,"abstract":"<p><p>The extraction of high-quality RNA from kenaf is essential for genetic and molecular biology research. However, the presence of high levels of polysaccharide and polyphenol compounds in kenaf poses challenges for RNA isolation. We proposed a simplified, time-saving and cost-effective method for isolating high quantities of RNA from various kenaf tissues. This method exhibited superior efficiency in RNA isolation compared with the conventional cetyltrimethylammonium bromide method and demonstrated greater adaptability to different samples than commercial kits. Furthermore, the high-quality RNA obtained from this method was successfully utilized for RT-PCR, real-time RT-PCR and northern blot analysis. Moreover, this proposed protocol also enables the acquisition of both high-quality and -quantity gDNA through RNase A treatment. In addition, the effectiveness of this approach in isolating high-quality RNA from other plant species has been experimentally confirmed.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"218-226"},"PeriodicalIF":2.7,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50160551","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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