Jing Xu, Pawan K Pandoh, Richard D Corbett, Duane Smailus, Reanne Bowlby, Denise Brooks, Helen McDonald, Simon Haile, Sundeep Chahal, Steve Bilobram, Karen L Mungall, Andrew J Mungall, Robin Coope, Richard A Moore, Yongjun Zhao, Steven Jm Jones, Marco A Marra
High-throughput total nucleic acid (TNA) purification methods based on solid-phase reversible immobilization (SPRI) beads produce TNA suitable for both genomic and transcriptomic applications. Even so, small RNA species, including miRNA, bind weakly to SPRI beads under standard TNA purification conditions, necessitating a separate workflow using column-based methods that are difficult to automate. Here, an SPRI-based high-throughput TNA purification protocol that recovers DNA, RNA and small RNA, called GSC-modified RLT+ Aline bead-based protocol (GRAB-ALL), which incorporates modifications to enhance small RNA recovery is presented. GRAB-ALL was benchmarked against existing nucleic acid purification workflows and GRAB-ALL efficiently purifies TNA, including small RNA, for next-generation sequencing applications in a plate-based format suitable for automated high-throughput sample preparation.
{"title":"A high-throughput pipeline for DNA/RNA/small RNA purification from tissue samples for sequencing.","authors":"Jing Xu, Pawan K Pandoh, Richard D Corbett, Duane Smailus, Reanne Bowlby, Denise Brooks, Helen McDonald, Simon Haile, Sundeep Chahal, Steve Bilobram, Karen L Mungall, Andrew J Mungall, Robin Coope, Richard A Moore, Yongjun Zhao, Steven Jm Jones, Marco A Marra","doi":"10.2144/btn-2023-0011","DOIUrl":"https://doi.org/10.2144/btn-2023-0011","url":null,"abstract":"<p><p>High-throughput total nucleic acid (TNA) purification methods based on solid-phase reversible immobilization (SPRI) beads produce TNA suitable for both genomic and transcriptomic applications. Even so, small RNA species, including miRNA, bind weakly to SPRI beads under standard TNA purification conditions, necessitating a separate workflow using column-based methods that are difficult to automate. Here, an SPRI-based high-throughput TNA purification protocol that recovers DNA, RNA and small RNA, called GSC-modified RLT+ Aline bead-based protocol (GRAB-ALL), which incorporates modifications to enhance small RNA recovery is presented. GRAB-ALL was benchmarked against existing nucleic acid purification workflows and GRAB-ALL efficiently purifies TNA, including small RNA, for next-generation sequencing applications in a plate-based format suitable for automated high-throughput sample preparation.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10058280","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}
Transfection, a nonviral method of nucleic acid delivery, often exhibits poor efficiency in vivo. The needle-based in vivo delivery of transfection reagents can be invasive. Here, we report a noninvasive protocol for in vivo gene delivery via the needle-free MED-JET H4 MULTIJET (MJH4M) device using both "home-made" glucose-based and commercial transfection reagents. The objective of this study was to compare the relative transfection efficiencies of the needle-free system to that of the needle-based delivery method. We observed a 15-fold increase in transfection efficiency using the needle-free MJH4M device when compared to the needle-based delivery method. The highest transfection efficiency was achieved using a 5% glucose solution as the delivery vehicle.
{"title":"A novel use of a needle-free injection system for improved nucleic acid delivery and expression <i>in vivo</i>.","authors":"Nathan Liu, Hani Abd-Ul-Salam, Noémie Joannette-Lafrance, Jingjing Li, Karim Menassa, Monzur Murshed","doi":"10.2144/btn-2023-0015","DOIUrl":"https://doi.org/10.2144/btn-2023-0015","url":null,"abstract":"<p><p>Transfection, a nonviral method of nucleic acid delivery, often exhibits poor efficiency <i>in vivo</i>. The needle-based <i>in vivo</i> delivery of transfection reagents can be invasive. Here, we report a noninvasive protocol for <i>in vivo</i> gene delivery via the needle-free MED-JET H4 MULTIJET (MJH4M) device using both \"home-made\" glucose-based and commercial transfection reagents. The objective of this study was to compare the relative transfection efficiencies of the needle-free system to that of the needle-based delivery method. We observed a 15-fold increase in transfection efficiency using the needle-free MJH4M device when compared to the needle-based delivery method. The highest transfection efficiency was achieved using a 5% glucose solution as the delivery vehicle.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10057766","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":"Chromatin immunoprecipitation and library preparation: a powerful tool to unravel the epigenome.","authors":"Mukesh Pratap Yadav, Vikas Kundra","doi":"10.2144/btn-2022-0118","DOIUrl":"https://doi.org/10.2144/btn-2022-0118","url":null,"abstract":"","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9882904","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}
Standfirst With an ever-growing global population, the pursuit of sustainable agriculture has become paramount. What if the solution lies right beneath our feet? Enter the root microbiome, the hidden hero poised to revolutionize agriculture and bring us closer to a greener future. [Formula: see text].
{"title":"The root microbiome: techniques for exploration and agricultural applications.","authors":"Ashling Cannon","doi":"10.2144/btn-2023-0057","DOIUrl":"https://doi.org/10.2144/btn-2023-0057","url":null,"abstract":"<p><p>Standfirst With an ever-growing global population, the pursuit of sustainable agriculture has become paramount. What if the solution lies right beneath our feet? Enter the root microbiome, the hidden hero poised to revolutionize agriculture and bring us closer to a greener future. [Formula: see text].</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9883899","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}
A PCR cloning method that combines a dual selection pGATE-1 plasmid vector and an improved overlap extension cloning was developed. This efficient and cost-effective method allows for the introduction of DNA fragments into the Gateway cloning pipeline. The cloning efficiency is facilitated by a dual selection that includes the ccdB gene and gentamicin resistance. For users of the Gateway cloning system, substantial cost saving comes from eliminating BP recombination and ligation reactions to introduce DNA fragments into pDONR or pENTR vectors. Beyond the Gateway technology, this recombination-based cloning system can be used to efficiently clone PCR amplicons by adding 24-base pair adaptor sequences that are utilized by bacterial homologous recombination mechanism.
{"title":"Overlap extension cloning of PCR products into a Gateway-compatible plasmid vector.","authors":"Omid Zare-Mehrjerdi, Gracie Trader, Viktor Kirik","doi":"10.2144/btn-2023-0001","DOIUrl":"https://doi.org/10.2144/btn-2023-0001","url":null,"abstract":"A PCR cloning method that combines a dual selection pGATE-1 plasmid vector and an improved overlap extension cloning was developed. This efficient and cost-effective method allows for the introduction of DNA fragments into the Gateway cloning pipeline. The cloning efficiency is facilitated by a dual selection that includes the ccdB gene and gentamicin resistance. For users of the Gateway cloning system, substantial cost saving comes from eliminating BP recombination and ligation reactions to introduce DNA fragments into pDONR or pENTR vectors. Beyond the Gateway technology, this recombination-based cloning system can be used to efficiently clone PCR amplicons by adding 24-base pair adaptor sequences that are utilized by bacterial homologous recombination mechanism.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/61/ea/btn-75-363.PMC10388215.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9972567","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}
Pub Date : 2023-07-01Epub Date: 2023-06-09DOI: 10.2144/btn-2023-0029
Christopher L Hemme, Rachel Carley, Arielle Norton, Moez Ghumman, Hannah Nguyen, Ryan Ivone, Jyothi U Menon, Jie Shen, Matthew Bertin, Roberta King, Elizabeth Leibovitz, Roy Bergstrom, Bongsup Cho
The Rhode Island IDeA Network of Biomedical Research Excellence Molecular Informatics Core at the University of Rhode Island Information Technology Services Innovative Learning Technologies developed virtual and augmented reality applications to teach concepts in biomedical science, including pharmacology, medicinal chemistry, cell culture and nanotechnology. The apps were developed as full virtual reality/augmented reality and 3D gaming versions, which do not require virtual reality headsets. Development challenges included creating intuitive user interfaces, text-to-voice functionality, visualization of molecules and implementing complex science concepts. In-app quizzes are used to assess the user's understanding of topics, and user feedback was collected for several apps to improve the experience. The apps were positively reviewed by users and are being implemented into the curriculum at the University of Rhode Island.
{"title":"Developing virtual and augmented reality applications for science, technology, engineering and math education.","authors":"Christopher L Hemme, Rachel Carley, Arielle Norton, Moez Ghumman, Hannah Nguyen, Ryan Ivone, Jyothi U Menon, Jie Shen, Matthew Bertin, Roberta King, Elizabeth Leibovitz, Roy Bergstrom, Bongsup Cho","doi":"10.2144/btn-2023-0029","DOIUrl":"10.2144/btn-2023-0029","url":null,"abstract":"<p><p>The Rhode Island IDeA Network of Biomedical Research Excellence Molecular Informatics Core at the University of Rhode Island Information Technology Services Innovative Learning Technologies developed virtual and augmented reality applications to teach concepts in biomedical science, including pharmacology, medicinal chemistry, cell culture and nanotechnology. The apps were developed as full virtual reality/augmented reality and 3D gaming versions, which do not require virtual reality headsets. Development challenges included creating intuitive user interfaces, text-to-voice functionality, visualization of molecules and implementing complex science concepts. In-app quizzes are used to assess the user's understanding of topics, and user feedback was collected for several apps to improve the experience. The apps were positively reviewed by users and are being implemented into the curriculum at the University of Rhode Island.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10505987/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10290714","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}
Quantitative bioanalysis is essential when establishing pharmacokinetic properties during the drug development process. To overcome challenges of sensitivity, specificity and process complexity associated with the conventional analysis of antisense oligonucleotides (ASOs), a new approach to nonenzymatic hybridization assays using probe alteration-linked self-assembly reaction (PALSAR) technology as a signal amplifier was evaluated. PALSAR quantification of ASOs in mouse tissue and plasma was able to achieve a high sensitivity ranging from 1.5 to 6 pg/ml, intra-/interday accuracies in the range of 86.8-119.1% and 88.1-113.1%, respectively, and precision of ≤17.2%. Furthermore, crossreactivity of 3'n-1, a metabolite with a single base difference, was <1%. Our approach provides an auspicious method for distinguishing metabolites and detecting ASOs with high sensitivity and specificity.
{"title":"Sensitive and specific quantification of antisense oligonucleotides using probe alteration-linked self-assembly reaction technology.","authors":"Masako Osawa, Takurou Akiya, Funa Ogawa, Takao Suzuki, Masaki Yamagami, Tadashi Umemoto, Akira Ideno","doi":"10.2144/btn-2023-0005","DOIUrl":"https://doi.org/10.2144/btn-2023-0005","url":null,"abstract":"<p><p>Quantitative bioanalysis is essential when establishing pharmacokinetic properties during the drug development process. To overcome challenges of sensitivity, specificity and process complexity associated with the conventional analysis of antisense oligonucleotides (ASOs), a new approach to nonenzymatic hybridization assays using probe alteration-linked self-assembly reaction (PALSAR) technology as a signal amplifier was evaluated. PALSAR quantification of ASOs in mouse tissue and plasma was able to achieve a high sensitivity ranging from 1.5 to 6 pg/ml, intra-/interday accuracies in the range of 86.8-119.1% and 88.1-113.1%, respectively, and precision of ≤17.2%. Furthermore, crossreactivity of 3'n-1, a metabolite with a single base difference, was <1%. Our approach provides an auspicious method for distinguishing metabolites and detecting ASOs with high sensitivity and specificity.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9946132","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}
Our planet faces many challenges. Namely, the damage caused by human disruption of natural levels of carbon and nitrogen as a result of the use of fossil fuels. Microbes play a crucial role in the biogeochemical processing of these elements, in turn placing them at the heart of one of the most testing issues of all time - climate change. [Formula: see text].
{"title":"Microbial marvels: could microbes resolve climate change?","authors":"Jade Parker","doi":"10.2144/btn-2023-0043","DOIUrl":"https://doi.org/10.2144/btn-2023-0043","url":null,"abstract":"<p><p>Our planet faces many challenges. Namely, the damage caused by human disruption of natural levels of carbon and nitrogen as a result of the use of fossil fuels. Microbes play a crucial role in the biogeochemical processing of these elements, in turn placing them at the heart of one of the most testing issues of all time - climate change. [Formula: see text].</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9927852","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}
Pub Date : 2023-06-01Epub Date: 2023-06-16DOI: 10.2144/btn-2023-0039
Himanshu Kaul
{"title":"Multiscale computational modeling offers key to understanding molecular logic underpinning development and disease.","authors":"Himanshu Kaul","doi":"10.2144/btn-2023-0039","DOIUrl":"10.2144/btn-2023-0039","url":null,"abstract":"","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9873857","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}
Secondary metabolites in mangroves often interfere with RNA extraction yielding poor concentration and quality, which is unsuitable for downstream applications. As existing protocols yielded low-quality RNA from root tissues of Kandelia candel (L.) Druce and Rhizophora mucronata Lam., an optimized method was developed for improving the quality and yield of RNA. Compared with three other methods, this optimized protocol gave better RNA yield and purity for both species. The absorbance ratios were ≥1.9 for A260/280 and A260/230, while RNA integrity number values ranged from 7.5 to 9.6. Results show that our modified method is efficient in obtaining high-quality RNA from mangrove roots and is suitable for downstream experiments such as cDNA synthesis, real-time quantitative PCR and next-generation sequencing.
{"title":"A modified method for efficient RNA isolation from mangrove root tissues rich in secondary metabolites.","authors":"Ashifa Nizam, Haritha Kalath, Ajay Kumar","doi":"10.2144/btn-2022-0078","DOIUrl":"https://doi.org/10.2144/btn-2022-0078","url":null,"abstract":"<p><p>Secondary metabolites in mangroves often interfere with RNA extraction yielding poor concentration and quality, which is unsuitable for downstream applications. As existing protocols yielded low-quality RNA from root tissues of <i>Kandelia candel</i> (L.) Druce and <i>Rhizophora mucronata</i> Lam., an optimized method was developed for improving the quality and yield of RNA. Compared with three other methods, this optimized protocol gave better RNA yield and purity for both species. The absorbance ratios were ≥1.9 for A260/280 and A260/230, while RNA integrity number values ranged from 7.5 to 9.6. Results show that our modified method is efficient in obtaining high-quality RNA from mangrove roots and is suitable for downstream experiments such as cDNA synthesis, real-time quantitative PCR and next-generation sequencing.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9873093","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}