Pub Date : 2024-05-22DOI: 10.1093/biomethods/bpae034
I. Fasogbon, Erick Nyakundi Ondari, Tusubira Deusdedit, Loganathan Rangasamy, Sasirekha Krishnan, P. M. Aja
Point-of-care (POC) field screening for tools for Mycoplasma bovis (M. bovis) is still lacking due to the requirement for a simple, robust field-applicable test that does not entail specialized laboratory equipment. In accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines, this review identifies the methodologies that were retrieved based on our search strategy, that have been reported for the diagnosis of m. bovis infection between 2014 and diagnostics. A search criteria was generated to curate one hundred and three articles, which were reduced in number (to 46), following the screening guidelines of PRISMA. The 43 articles included in the study present twenty-five different assay methods. The assay methods were grouped as microbiological culture, serological assay, PCR-based assay, LAMP-based assay, NGS-based assay, or Lateral flow assay. We, however, focus our discussion on the three lateral flow-based assays relative to others, highlighting the advantages they present above the other techniques and their potential applicability as a POC diagnostic test for M. bovis infections. We therefore call for further research on developing a lateral flow-based screening tool that could revolutionize the diagnosis of M. bovis infection.
{"title":"Point-of-care potentials of lateral flow-based field screening for MYCOPLASMA BOVIS infections: a literature review","authors":"I. Fasogbon, Erick Nyakundi Ondari, Tusubira Deusdedit, Loganathan Rangasamy, Sasirekha Krishnan, P. M. Aja","doi":"10.1093/biomethods/bpae034","DOIUrl":"https://doi.org/10.1093/biomethods/bpae034","url":null,"abstract":"\u0000 Point-of-care (POC) field screening for tools for Mycoplasma bovis (M. bovis) is still lacking due to the requirement for a simple, robust field-applicable test that does not entail specialized laboratory equipment. In accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines, this review identifies the methodologies that were retrieved based on our search strategy, that have been reported for the diagnosis of m. bovis infection between 2014 and diagnostics. A search criteria was generated to curate one hundred and three articles, which were reduced in number (to 46), following the screening guidelines of PRISMA. The 43 articles included in the study present twenty-five different assay methods. The assay methods were grouped as microbiological culture, serological assay, PCR-based assay, LAMP-based assay, NGS-based assay, or Lateral flow assay. We, however, focus our discussion on the three lateral flow-based assays relative to others, highlighting the advantages they present above the other techniques and their potential applicability as a POC diagnostic test for M. bovis infections. We therefore call for further research on developing a lateral flow-based screening tool that could revolutionize the diagnosis of M. bovis infection.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141112391","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-22DOI: 10.1093/biomethods/bpae038
Saran Lotfollahzadeh, Asha Jose, Esha Shafiq zarnaab, Nourhan El Sherif, Michael Smith, Jingyan Han, Francesca Seta, V. Chitalia
Vascular smooth muscle cells (VSMCs) are an integral part of blood vessels and are the focus of intensive research in vascular biology, translational research, and cardiovascular diseases. Though immortalized vascular smooth muscle cell lines are available, their use is limited, underscoring the need for primary VSMCs. There are several methods for isolating primary cells from mice. However, the isolation method from rat blood vessels requires optimization, given the differences in the aorta of mice and rats. Here we compare two methods for VSMCs isolation from rats: enzymatic digestion and the “block” method. We observed a significantly higher yield of VSMCs using the enzymatic digestion method. We further confirmed that VSMCs expressed well-established VSMC-specific markers (calponin) with both methods and observed the persistence of this marker up to 9 passages, suggesting a continuation of the secretory phenotype of VSMCs. Overall, this work compares two methods and demonstrates a practical and effective method for isolating VSMCs from rat aorta, providing vascular biologists with a valuable and reliable experimental tool.
{"title":"Two methods of isolation of rat aortic smooth muscle cells with high yield","authors":"Saran Lotfollahzadeh, Asha Jose, Esha Shafiq zarnaab, Nourhan El Sherif, Michael Smith, Jingyan Han, Francesca Seta, V. Chitalia","doi":"10.1093/biomethods/bpae038","DOIUrl":"https://doi.org/10.1093/biomethods/bpae038","url":null,"abstract":"\u0000 Vascular smooth muscle cells (VSMCs) are an integral part of blood vessels and are the focus of intensive research in vascular biology, translational research, and cardiovascular diseases. Though immortalized vascular smooth muscle cell lines are available, their use is limited, underscoring the need for primary VSMCs. There are several methods for isolating primary cells from mice. However, the isolation method from rat blood vessels requires optimization, given the differences in the aorta of mice and rats. Here we compare two methods for VSMCs isolation from rats: enzymatic digestion and the “block” method. We observed a significantly higher yield of VSMCs using the enzymatic digestion method. We further confirmed that VSMCs expressed well-established VSMC-specific markers (calponin) with both methods and observed the persistence of this marker up to 9 passages, suggesting a continuation of the secretory phenotype of VSMCs. Overall, this work compares two methods and demonstrates a practical and effective method for isolating VSMCs from rat aorta, providing vascular biologists with a valuable and reliable experimental tool.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141112957","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-17DOI: 10.1093/biomethods/bpae033
Zixian Li, Mia Bilic, Bhushan Nagar
Visualizing RNA-protein interactions through structural approaches requires the use of RNA molecules purified to homogeneity. We describe here a simple and effective method, free of acrylamide contamination and without using UV radiation, to separate in vitro synthesized, heterogeneous RNA transcripts (up to ∼15 nucleotides) at single nucleotide resolution by quaternary-amine anion exchange chromatography. The quality of short RNAs isolated through this method is validated by gel electrophoresis, mass spectrometry, and crystallization with a protein binding partner.
{"title":"Isolation of short RNAs with homogenous 3’-ends using quaternary-amine anion exchange chromatography","authors":"Zixian Li, Mia Bilic, Bhushan Nagar","doi":"10.1093/biomethods/bpae033","DOIUrl":"https://doi.org/10.1093/biomethods/bpae033","url":null,"abstract":"\u0000 Visualizing RNA-protein interactions through structural approaches requires the use of RNA molecules purified to homogeneity. We describe here a simple and effective method, free of acrylamide contamination and without using UV radiation, to separate in vitro synthesized, heterogeneous RNA transcripts (up to ∼15 nucleotides) at single nucleotide resolution by quaternary-amine anion exchange chromatography. The quality of short RNAs isolated through this method is validated by gel electrophoresis, mass spectrometry, and crystallization with a protein binding partner.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-05-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140964356","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-17DOI: 10.1093/biomethods/bpae032
Ramin Khanabdali, Michelle Mandrekar, Rick Grygiel, Phuoc-an Vo, Carlos Palma, Sara Nikseresht, Siena Barton, Mozhgan Shojaee, Sadman Bhuiyan, Kartini Asari, Susan Belzer, Khairul Ansari, Jermaine I Coward, Lewis Perrin, John Hooper, Dominic Guanzon, Andrew Lai, Carlos Salomon, Kevin Kershner, Christine Newton, Douglas Horejsh, Gregory E. Rice
Extracellular vesicles (EVs), including exosomes, have significant potential for diagnostic and therapeutic applications. The lack of standardized methods for efficient and high throughput isolation and analysis of EVs, however, has limited their widespread use in clinical practice. Surface epitope immunoaffinity (SEI) isolation utilises affinity ligands, including antibodies, aptamers, or lectins, that target specific surface proteins present on EVs. Paramagnetic bead-SEI isolation represents a fit-for-purpose solution for the reproducible, high throughput isolation of EVs from biofluids and downstream analysis of RNA, protein and lipid biomarkers that is compatible with clinical laboratory workflows. This study evaluates a new surface epitope immunoaffinity isolation method for enriching subpopulations of EVs. EVs were isolated from human plasma using a bead-based SEI method designed for on-bead and downstream analysis of EV-associated RNA and protein biomarkers. Western blot analysis confirmed the presence of EV markers in the captured nanoparticles. Mass spectrometry analysis of the SEI lysate identified over 1500 proteins, with the top 100 including known EV-associated proteins. miRNA sequencing followed by RT-qPCR analysis identified EV-associated miRNA transcripts. Using SEI, EVs were isolated using automated high throughput particle moving instruments, demonstrating equal or higher protein and miRNA yield and recovery compared to manual processing. SEI is a rapid, efficient, and high throughput method for isolating enriched populations of EVs; effectively reducing contamination and enabling the isolation of a specific subpopulation of EVs. In this study, high throughput EV isolation and RNA extraction have been successfully implemented. This technology holds great promise for advancing the field of EV research and facilitating their application for biomarker discovery and clinical research.
包括外泌体在内的细胞外囊泡（EVs）在诊断和治疗方面具有巨大的应用潜力。然而，由于缺乏高效、高通量分离和分析 EVs 的标准化方法，限制了它们在临床实践中的广泛应用。表面表位免疫亲和（SEI）分离利用的是亲和配体，包括抗体、适配体或凝集素，它们针对的是存在于 EVs 上的特定表面蛋白。顺磁珠-SEI 分离是一种适用于从生物流体中可重复、高通量分离 EVs 并进行下游 RNA、蛋白质和脂质生物标记物分析的解决方案，与临床实验室工作流程兼容。本研究评估了一种用于富集 EVs 亚群的新型表面表位免疫亲和分离方法。使用一种基于珠子的 SEI 方法从人血浆中分离出了 EVs，该方法设计用于珠子上和下游的 EV 相关 RNA 和蛋白质生物标记物分析。Western 印迹分析证实了捕获的纳米颗粒中存在 EV 标记。SEI 裂解液的质谱分析鉴定了超过 1500 种蛋白质，其中前 100 种包括已知的 EV 相关蛋白质。利用 SEI，使用自动高通量粒子移动仪器分离了 EV，与人工处理相比，蛋白质和 miRNA 的产量和回收率相同或更高。SEI 是一种快速、高效、高通量的方法，可用于分离富集的 EVs 群体；有效减少污染，并能分离特定的 EVs 亚群。本研究成功实现了高通量 EV 分离和 RNA 提取。这项技术有望推动 EV 研究领域的发展，并促进其在生物标记物发现和临床研究中的应用。
{"title":"High throughput Surface Epitope Immunoaffinity Isolation of Extracellular Vesicles and Downstream Analysis","authors":"Ramin Khanabdali, Michelle Mandrekar, Rick Grygiel, Phuoc-an Vo, Carlos Palma, Sara Nikseresht, Siena Barton, Mozhgan Shojaee, Sadman Bhuiyan, Kartini Asari, Susan Belzer, Khairul Ansari, Jermaine I Coward, Lewis Perrin, John Hooper, Dominic Guanzon, Andrew Lai, Carlos Salomon, Kevin Kershner, Christine Newton, Douglas Horejsh, Gregory E. Rice","doi":"10.1093/biomethods/bpae032","DOIUrl":"https://doi.org/10.1093/biomethods/bpae032","url":null,"abstract":"\u0000 Extracellular vesicles (EVs), including exosomes, have significant potential for diagnostic and therapeutic applications. The lack of standardized methods for efficient and high throughput isolation and analysis of EVs, however, has limited their widespread use in clinical practice. Surface epitope immunoaffinity (SEI) isolation utilises affinity ligands, including antibodies, aptamers, or lectins, that target specific surface proteins present on EVs. Paramagnetic bead-SEI isolation represents a fit-for-purpose solution for the reproducible, high throughput isolation of EVs from biofluids and downstream analysis of RNA, protein and lipid biomarkers that is compatible with clinical laboratory workflows. This study evaluates a new surface epitope immunoaffinity isolation method for enriching subpopulations of EVs. EVs were isolated from human plasma using a bead-based SEI method designed for on-bead and downstream analysis of EV-associated RNA and protein biomarkers. Western blot analysis confirmed the presence of EV markers in the captured nanoparticles. Mass spectrometry analysis of the SEI lysate identified over 1500 proteins, with the top 100 including known EV-associated proteins. miRNA sequencing followed by RT-qPCR analysis identified EV-associated miRNA transcripts. Using SEI, EVs were isolated using automated high throughput particle moving instruments, demonstrating equal or higher protein and miRNA yield and recovery compared to manual processing. SEI is a rapid, efficient, and high throughput method for isolating enriched populations of EVs; effectively reducing contamination and enabling the isolation of a specific subpopulation of EVs. In this study, high throughput EV isolation and RNA extraction have been successfully implemented. This technology holds great promise for advancing the field of EV research and facilitating their application for biomarker discovery and clinical research.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-05-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140964328","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-17DOI: 10.1093/biomethods/bpae031
Bernhard Gill, Theresa Kehler, Michael Schneider
Determining "excess mortality" makes it possible to compare the burden of disasters between countries and over time, and thus also to evaluate the success of mitigation measures. However, the debate on Covid-19 has exposed that calculations of excess mortalities vary considerably depending on the method and its specification. Moreover, it is often unclear what exactly is meant by "excess mortality". We define excess mortality as the excess over the number of deaths that would have been expected counter-factually, ie without the catastrophic event in question. Based on this definition, we use a very parsimonious calculation method, namely the linear extrapolation of death figures from previous years to determine the excess mortality during the Covid-19 pandemic. But unlike most other literature on this topic, we first evaluated and optimised the specification of our method using a larger historical data set in order to identify and minimise estimation errors and biases. The result shows that excess mortality rates in the literature are often inflated. Moreover, they would have exhibited considerable excess mortalities in the period before Covid-19, if this value had already been of public interest at that time. Three conclusions can be drawn from this study and its findings: 1) All calculation methods for current figures should first be evaluated against past figures. 2) To avoid alarm fatigue, for mass media and policy communication thresholds should be introduced which would differentiate between "usual fluctuations" and "remarkable excess". 3) Statistical offices could provide more realistic estimates.
{"title":"Meaning and prediction of \"excess mortality\": A comparison of Covid- and pre-Covid mortality data in 31 Eurostat countries from 1965 to 2021","authors":"Bernhard Gill, Theresa Kehler, Michael Schneider","doi":"10.1093/biomethods/bpae031","DOIUrl":"https://doi.org/10.1093/biomethods/bpae031","url":null,"abstract":"\u0000 Determining \"excess mortality\" makes it possible to compare the burden of disasters between countries and over time, and thus also to evaluate the success of mitigation measures. However, the debate on Covid-19 has exposed that calculations of excess mortalities vary considerably depending on the method and its specification. Moreover, it is often unclear what exactly is meant by \"excess mortality\". We define excess mortality as the excess over the number of deaths that would have been expected counter-factually, ie without the catastrophic event in question. Based on this definition, we use a very parsimonious calculation method, namely the linear extrapolation of death figures from previous years to determine the excess mortality during the Covid-19 pandemic. But unlike most other literature on this topic, we first evaluated and optimised the specification of our method using a larger historical data set in order to identify and minimise estimation errors and biases. The result shows that excess mortality rates in the literature are often inflated. Moreover, they would have exhibited considerable excess mortalities in the period before Covid-19, if this value had already been of public interest at that time. Three conclusions can be drawn from this study and its findings: 1) All calculation methods for current figures should first be evaluated against past figures. 2) To avoid alarm fatigue, for mass media and policy communication thresholds should be introduced which would differentiate between \"usual fluctuations\" and \"remarkable excess\". 3) Statistical offices could provide more realistic estimates.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-05-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140962652","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-16DOI: 10.1093/biomethods/bpae030
Travis A Rainey, Emily E Tryc, Kirsten E Nicholson
Multiple methods for collecting genetic samples from amphibians exist, each with their own implications for study design, animal welfare, and costs. Toe clipping is one common method, but there is ongoing debate regarding its potential detriment. Less invasive methods should be implemented, if efficacious, as amphibians are a particularly vulnerable vertebrate group. Skin and buccal swabbing are less invasive methods for genetic sampling, but the potential for contamination and a lower yield of DNA may exist. To compare these methods, we gathered skin swabs, buccal swabs, and toe clips from the same individuals of a relatively small anuran species, Blanchard’s Cricket Frog (Acris blanchardi). We then compared DNA yield, DNA purity, amplification success rate, and genotypic data quality among sample types. We found toe clips and buccal swabs generated similar DNA yield and purity, with skin swabs yielding significantly less DNA of significantly lower purity than the other sample types. Amplification success rate was significantly higher using toe clips compared to the other sample types, though buccal swab samples amplified more readily than skin swabs. Genotypic data from toe clips and buccal swabs did not differ significantly in quality, but skin swab data quality was significantly lowest among sample types. Thus, skin swabbing could produce erroneous data in some situations, but buccal swabbing is likely an effective substitute to toe clipping, even for small species. Our results can help future researchers select which genetic sampling method might best suit their research needs.
从两栖动物身上采集基因样本有多种方法，每种方法对研究设计、动物福利和成本都有各自的影响。剪趾是一种常见的方法，但关于这种方法的潜在危害一直存在争议。由于两栖动物是特别脆弱的脊椎动物群体，因此如果有效，应采用侵入性较小的方法。皮肤和颊拭子是侵入性较小的基因采样方法，但可能存在污染和 DNA 产量较低的可能性。为了比较这些方法，我们收集了皮肤拭子、口腔拭子和脚趾夹，这些拭子取自相对较小的无脊椎动物物种--布兰查德蟋蟀蛙（Acris blanchardi）的相同个体。然后，我们比较了不同样本类型的 DNA 产量、DNA 纯度、扩增成功率和基因型数据质量。我们发现脚趾夹和颊拭子产生的DNA产量和纯度相似，而皮肤拭子产生的DNA产量和纯度明显低于其他类型的样本。与其他样本类型相比，脚趾夹的扩增成功率明显更高，但颊拭子样本比皮肤拭子更容易扩增。趾夹和口腔拭子的基因型数据在质量上没有明显差异，但皮肤拭子的数据质量在所有样本类型中明显最低。因此，在某些情况下，皮肤拭子可能会产生错误的数据，但口腔拭子可能是剪趾的有效替代品，即使对于小型物种也是如此。我们的研究结果可以帮助未来的研究人员选择最适合其研究需要的基因采样方法。
{"title":"Comparing skin swabs, buccal swabs, and toe clips for amphibian genetic sampling, a case study with a small anuran (acris blanchardi)","authors":"Travis A Rainey, Emily E Tryc, Kirsten E Nicholson","doi":"10.1093/biomethods/bpae030","DOIUrl":"https://doi.org/10.1093/biomethods/bpae030","url":null,"abstract":"\u0000 Multiple methods for collecting genetic samples from amphibians exist, each with their own implications for study design, animal welfare, and costs. Toe clipping is one common method, but there is ongoing debate regarding its potential detriment. Less invasive methods should be implemented, if efficacious, as amphibians are a particularly vulnerable vertebrate group. Skin and buccal swabbing are less invasive methods for genetic sampling, but the potential for contamination and a lower yield of DNA may exist. To compare these methods, we gathered skin swabs, buccal swabs, and toe clips from the same individuals of a relatively small anuran species, Blanchard’s Cricket Frog (Acris blanchardi). We then compared DNA yield, DNA purity, amplification success rate, and genotypic data quality among sample types. We found toe clips and buccal swabs generated similar DNA yield and purity, with skin swabs yielding significantly less DNA of significantly lower purity than the other sample types. Amplification success rate was significantly higher using toe clips compared to the other sample types, though buccal swab samples amplified more readily than skin swabs. Genotypic data from toe clips and buccal swabs did not differ significantly in quality, but skin swab data quality was significantly lowest among sample types. Thus, skin swabbing could produce erroneous data in some situations, but buccal swabbing is likely an effective substitute to toe clipping, even for small species. Our results can help future researchers select which genetic sampling method might best suit their research needs.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-05-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140967873","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-06DOI: 10.1093/biomethods/bpae029
Lin Chen, Caroline Bosmajian, Sukyung Woo
Small interfering RNA (siRNA) is a powerful tool for sequence-specific silencing of disease-related genes. In this study, we established and validated a stem-loop reverse transcription real-time polymerase chain reaction (RT-qPCR) method applicable for both chemically unmodified and modified siRNA, aiming to elucidate mechanistic intracellular pharmacokinetic and pharmacodynamic (PK/PD) properties of siRNA. We conducted a comprehensive evaluation of factors affecting intracellular siRNA quantification. Our study revealed that immobilization-based siRNA extraction introduced high variation, making it unsuitable for absolute quantification. Conversely, direct cell lysis followed by stem-loop RT-qPCR demonstrated excellent reproducibility, with a quantification range from 0.0002 to 20 femtomole (fmole) for unmodified siRNA and 0.02 to 20 fmole for modified siRNA. The design of a 6-basepair overlapping RT primer facilitated the distinction of full-length antisense from its 3’ metabolites, and pre-annealing of antisense to RT primer enhanced sensitivity and reproducibility. Differences in siRNA loss during storage and sample processing were noted among microcentrifuge tubes from various manufacturers. Endogenous miR-16 served as a reference for normalizing cytoplasmic siRNA, while protein concentration post-immunoprecipitation lysis was used to normalize RISC-loaded siRNA levels. This method successfully enabled a detailed characterization of the time profiles of cytoplasmic and RISC-loaded siRNA, advancing of the in vitro-in vivo translation of siRNA therapeutics.
{"title":"A highly sensitive stem-loop RT-qPCR method to study siRNA intracellular pharmacokinetics and pharmacodynamics","authors":"Lin Chen, Caroline Bosmajian, Sukyung Woo","doi":"10.1093/biomethods/bpae029","DOIUrl":"https://doi.org/10.1093/biomethods/bpae029","url":null,"abstract":"\u0000 Small interfering RNA (siRNA) is a powerful tool for sequence-specific silencing of disease-related genes. In this study, we established and validated a stem-loop reverse transcription real-time polymerase chain reaction (RT-qPCR) method applicable for both chemically unmodified and modified siRNA, aiming to elucidate mechanistic intracellular pharmacokinetic and pharmacodynamic (PK/PD) properties of siRNA. We conducted a comprehensive evaluation of factors affecting intracellular siRNA quantification. Our study revealed that immobilization-based siRNA extraction introduced high variation, making it unsuitable for absolute quantification. Conversely, direct cell lysis followed by stem-loop RT-qPCR demonstrated excellent reproducibility, with a quantification range from 0.0002 to 20 femtomole (fmole) for unmodified siRNA and 0.02 to 20 fmole for modified siRNA. The design of a 6-basepair overlapping RT primer facilitated the distinction of full-length antisense from its 3’ metabolites, and pre-annealing of antisense to RT primer enhanced sensitivity and reproducibility. Differences in siRNA loss during storage and sample processing were noted among microcentrifuge tubes from various manufacturers. Endogenous miR-16 served as a reference for normalizing cytoplasmic siRNA, while protein concentration post-immunoprecipitation lysis was used to normalize RISC-loaded siRNA levels. This method successfully enabled a detailed characterization of the time profiles of cytoplasmic and RISC-loaded siRNA, advancing of the in vitro-in vivo translation of siRNA therapeutics.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141010220","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-04-27DOI: 10.1093/biomethods/bpae026
Mikaela M. Mallin, Louis T A Rolle, Kenneth J Pienta, S. Amend
Rapid and reliable circulating tumor cell (CTC) and disseminated tumor cell (DTC) detection is critical for rigorous evaluation of in vivo metastasis models. Clinical data shows that each step of the metastatic cascade presents increasing barriers to success, limiting the number of successful metastatic cells to fewer than 1 in 1,500,000,000. As such, it is critical for scientists to employ approaches that allow for evaluation of metastatic competency at each step of the cascade. Here, we present a flow cytometry-based method that enables swift and simultaneous comparison of both CTCs and DTCs from single animals, enabling evaluation of multiple metastatic steps within a single model system. We present the necessary gating strategy and optimized sample preparation conditions necessary to capture CTCs and DTCs using this approach. We also provide proof-of-concept experiments emphasizing the appropriate limits of detection of these conditions. Most importantly, we successfully recover CTCs and DTCs from murine blood and bone marrow. In supplemental materials, we expand the applicability of our method to lung tissue and exemplify a potential multi-plexing strategy to further characterize recovered CTCs and DTCs. This approach to multiparameter flow cytometric detection and analysis of rare cells in in vivo models of metastasis is reproducible, high-throughput, broadly applicable and highly adaptable to a wide range of scientific inquiries. Most notably, it simplifies the recovery and analysis of CTCs and DTCs from the same animal, allowing for a rapid first look at the comparative metastatic competency of various model systems throughout multiple steps of the metastatic cascade.
{"title":"Multiparameter flow cytometric detection and analysis of rare cells in in vivo models of cancer metastasis","authors":"Mikaela M. Mallin, Louis T A Rolle, Kenneth J Pienta, S. Amend","doi":"10.1093/biomethods/bpae026","DOIUrl":"https://doi.org/10.1093/biomethods/bpae026","url":null,"abstract":"\u0000 Rapid and reliable circulating tumor cell (CTC) and disseminated tumor cell (DTC) detection is critical for rigorous evaluation of in vivo metastasis models. Clinical data shows that each step of the metastatic cascade presents increasing barriers to success, limiting the number of successful metastatic cells to fewer than 1 in 1,500,000,000. As such, it is critical for scientists to employ approaches that allow for evaluation of metastatic competency at each step of the cascade. Here, we present a flow cytometry-based method that enables swift and simultaneous comparison of both CTCs and DTCs from single animals, enabling evaluation of multiple metastatic steps within a single model system. We present the necessary gating strategy and optimized sample preparation conditions necessary to capture CTCs and DTCs using this approach. We also provide proof-of-concept experiments emphasizing the appropriate limits of detection of these conditions. Most importantly, we successfully recover CTCs and DTCs from murine blood and bone marrow. In supplemental materials, we expand the applicability of our method to lung tissue and exemplify a potential multi-plexing strategy to further characterize recovered CTCs and DTCs. This approach to multiparameter flow cytometric detection and analysis of rare cells in in vivo models of metastasis is reproducible, high-throughput, broadly applicable and highly adaptable to a wide range of scientific inquiries. Most notably, it simplifies the recovery and analysis of CTCs and DTCs from the same animal, allowing for a rapid first look at the comparative metastatic competency of various model systems throughout multiple steps of the metastatic cascade.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140652041","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-04-27eCollection Date: 2024-01-01DOI: 10.1093/biomethods/bpae027
Julian Kammerer, Alexandra Cirnu, Tatjana Williams, Melanie Hasselmeier, Mike Nörpel, Ruping Chen, Brenda Gerull
Picrosirius red staining constitutes an important and broadly used tool to visualize collagen and fibrosis in various tissues. Although multiple qualitative and quantitative analysis methods to evaluate fibrosis are available, many require specialized devices and software or lack objectivity and scalability. Here, we aimed to develop a versatile and powerful "QuantSeg" macro in the FIJI image processing software capable of automated, robust, and quick collagen quantification in cardiac tissue from light micrographs. To examine different patterns of fibrosis, an optional segmentation algorithm was implemented. To ensure the method's validity, we quantified the collagen content in a set of wild-type versus plakoglobin-knockout murine hearts exhibiting extensive fibrosis using both the macro and an established, fluorescence microscopy-based method, and compared results. To demonstrate the capabilities of the segmentation feature, rat hearts were examined post-myocardial infarction. We found the QuantSeg macro to robustly detect the differences in fibrosis between knockout and control hearts. In sections with low collagen content, the macro yielded more consistent results than using the fluorescence microscopy-based technique. With its wide range of output parameters, ease of use, cost effectiveness, and objectivity, the QuantSeg macro has the potential to become an established method for analysis of PSR-stained tissue. The novel segmentation feature allows for automated evaluation of different patterns of cardiac fibrosis for the first time.
毕赤染色是观察各种组织中胶原蛋白和纤维化的一种重要且广泛使用的工具。虽然有多种定性和定量分析方法可用于评估纤维化，但许多方法都需要专门的设备和软件，或者缺乏客观性和可扩展性。在这里，我们的目标是在 FIJI 图像处理软件中开发一个多功能且功能强大的 "QuantSeg "宏，该宏能够自动、稳健、快速地从光显微照片中量化心脏组织中的胶原蛋白。为了检查不同的纤维化模式，我们采用了一种可选的分割算法。为确保该方法的有效性，我们使用宏法和一种成熟的基于荧光显微镜的方法量化了一组表现出广泛纤维化的野生型和plakoglobin-knockout小鼠心脏中的胶原蛋白含量，并对结果进行了比较。为了证明分割功能的能力，我们对心肌梗塞后的大鼠心脏进行了检查。我们发现，QuantSeg 宏能稳健地检测出基因敲除心脏和对照心脏纤维化的差异。在胶原蛋白含量较低的切片中，宏得到的结果比使用基于荧光显微镜的技术更一致。QuantSeg 宏有广泛的输出参数、易用性、成本效益和客观性，有望成为分析 PSR 染色组织的成熟方法。新颖的分割功能首次实现了对心脏纤维化不同模式的自动评估。
{"title":"Macro-based collagen quantification and segmentation in picrosirius red-stained heart sections using light microscopy.","authors":"Julian Kammerer, Alexandra Cirnu, Tatjana Williams, Melanie Hasselmeier, Mike Nörpel, Ruping Chen, Brenda Gerull","doi":"10.1093/biomethods/bpae027","DOIUrl":"10.1093/biomethods/bpae027","url":null,"abstract":"<p><p>Picrosirius red staining constitutes an important and broadly used tool to visualize collagen and fibrosis in various tissues. Although multiple qualitative and quantitative analysis methods to evaluate fibrosis are available, many require specialized devices and software or lack objectivity and scalability. Here, we aimed to develop a versatile and powerful \"<i>QuantSeg</i>\" macro in the FIJI image processing software capable of automated, robust, and quick collagen quantification in cardiac tissue from light micrographs. To examine different patterns of fibrosis, an optional segmentation algorithm was implemented. To ensure the method's validity, we quantified the collagen content in a set of wild-type versus plakoglobin-knockout murine hearts exhibiting extensive fibrosis using both the macro and an established, fluorescence microscopy-based method, and compared results. To demonstrate the capabilities of the segmentation feature, rat hearts were examined post-myocardial infarction. We found the <i>QuantSeg</i> macro to robustly detect the differences in fibrosis between knockout and control hearts. In sections with low collagen content, the macro yielded more consistent results than using the fluorescence microscopy-based technique. With its wide range of output parameters, ease of use, cost effectiveness, and objectivity, the <i>QuantSeg</i> macro has the potential to become an established method for analysis of PSR-stained tissue. The novel segmentation feature allows for automated evaluation of different patterns of cardiac fibrosis for the first time.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11116823/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141155716","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-04-26eCollection Date: 2024-01-01DOI: 10.1093/biomethods/bpae025
[This corrects the article DOI: 10.1093/biomethods/bpae015.].
[此处更正了文章 DOI：10.1093/biomethods/bpae015]。
{"title":"Correction to: An improved method for measuring catalase activity in biological samples.","authors":"","doi":"10.1093/biomethods/bpae025","DOIUrl":"https://doi.org/10.1093/biomethods/bpae025","url":null,"abstract":"<p><p>[This corrects the article DOI: 10.1093/biomethods/bpae015.].</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11052656/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140869672","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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