Methods in molecular biology最新文献_第9页

Assessing Amplification Quality and Bias in MDA Methods Through Comparative Analysis of Short-Read Sequencing. 通过短读段测序的比较分析评估MDA方法的扩增质量和偏倚。

Methods in molecular biology Pub Date : 2026-01-01 DOI: 10.1007/978-1-0716-5060-8_10

E D Lozano-Escobar, V Mateo-Cáceres, C Mayoral-Campos, M Redrejo-Rodríguez

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Sequencing RSV Whole Genome Using a Long Amplicon-Based Method with Oxford Nanopore Technologies. 利用牛津纳米孔技术对RSV全基因组进行长扩增子测序。

Methods in molecular biology Pub Date : 2026-01-01 DOI: 10.1007/978-1-0716-5060-8_11

Xiaomin Dong, Steven Edwards, Yi-Mo Deng, Clyde Dapat, Arada Hirankitti, Ian G Barr

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Whole-Genome Amplification of Single Circulating Tumor Cells from Mice Xenografts. 小鼠异种移植肿瘤循环细胞的全基因组扩增。

Methods in molecular biology Pub Date : 2026-01-01 DOI: 10.1007/978-1-0716-5060-8_5

Nuria Estévez-Gómez, Cristóbal Fernández-Santiago, Roberto Piñeiro, David Posada

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Application of Fourier Transform Infrared Spectroscopy for Structural Analysis of RNAs. 傅里叶变换红外光谱在rna结构分析中的应用。

Methods in molecular biology Pub Date : 2026-01-01 DOI: 10.1007/978-1-0716-5084-4_15

Frédéric Geinguenaud, Elsa Balduzzi, Véronique Arluison

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Laser Microdissection and Near Single-Cell Whole Genome Amplification. 激光显微解剖和近单细胞全基因组扩增。

Methods in molecular biology Pub Date : 2026-01-01 DOI: 10.1007/978-1-0716-5060-8_12

Roberto Cruz-Flores, Jorge Cáceres-Martínez, Arun K Dhar

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Semi-quantitative RT-PCR Assay for the Analysis of Alternative Splicing of Interleukin Genes. 白细胞介素基因选择性剪接的半定量RT-PCR分析。

Methods in molecular biology Pub Date : 2026-01-01 DOI: 10.1007/978-1-0716-4901-5_20

Md Mostafizur Rahman, Willy Munyao, Daisy Rubio, Shangwen Yan, Ariana Badalov, Christopher Beauvil, Nitya Sharma, Amatun Noor Prapty, Matteo Ruggiu

{"title":"Semi-quantitative RT-PCR Assay for the Analysis of Alternative Splicing of Interleukin Genes.","authors":"Md Mostafizur Rahman, Willy Munyao, Daisy Rubio, Shangwen Yan, Ariana Badalov, Christopher Beauvil, Nitya Sharma, Amatun Noor Prapty, Matteo Ruggiu","doi":"10.1007/978-1-0716-4901-5_20","DOIUrl":"https://doi.org/10.1007/978-1-0716-4901-5_20","url":null,"abstract":"Alternative splicing is a crucial post-transcriptional regulatory mechanism that generates multiple protein isoforms from a single gene, substantially increasing the coding capacity and proteome diversity of a genome. This process is particularly critical in regulating the activity of interleukin genes, where alternative splicing contributes to the functional diversity of these important immune system molecules and affects their production, function, and receptor interactions. While numerous studies have established the connection between aberrant alternative splicing and various diseases, including cancers and autoimmune disorders, the function and regulation of many splice variants remain poorly understood. Here, we describe a cost-effective and reliable method for analyzing alternative splicing patterns in interleukin genes using semi-quantitative RT-PCR and densitometry analysis. This method enables the simultaneous identification and quantification of multiple splice variants in a single PCR reaction, offering advantages over real-time RT-PCR approaches that require specific primer sets for each variant. This protocol involves RNA extraction from tissue culture cell lines or tissue samples, reverse transcription, RT-PCR, and subsequent analysis using freely available software for densitometry. We demonstrate the utility of this approach through two distinct examples with different alternative splicing patterns. While less sensitive than real-time RT-PCR or radioactive methods, this technique provides a robust, accessible, and widely accepted approach for investigating alternative splicing patterns in interleukin genes, contributing to our understanding of cytokine biology and its role in health and disease.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2983 ","pages":"231-248"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145889621","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}

引用次数: 0

Guidelines for the Design of Custom Affinity-Based Probes for Metalloproteases. 金属蛋白酶定制亲和探针设计指南。

Methods in molecular biology Pub Date : 2026-01-01 DOI: 10.1007/978-1-0716-5092-9_11

Marie Launay, Lomane Berthy, Sandra Llamas Rizo, Myrto Athina Balachouti, Dimitris Georgiadis, Laurent Devel

引用次数: 0

Application of Ultraviolet (UV) and Circular Dichroism (CD) Spectroscopies to Study RNA-RNA Interactions of Regulatory RNAs of Bacteriophage Origin. 应用紫外（UV）和圆二色（CD）光谱研究噬菌体起源调控rna的RNA-RNA相互作用。

Methods in molecular biology Pub Date : 2026-01-01 DOI: 10.1007/978-1-0716-5084-4_8

Natalia Lewandowska, Sylwia Bloch, Agnieszka Chylewska, Aleksandra M Dąbrowska, Monika Mazur, Joanna Zwolenkiewicz, Wojciech Wesołowski, Aleksandra Łukasiak, Emilia Węglińska, Mikołaj Olejniczak, Grzegorz Węgrzyn, Bożena Nejman-Faleńczyk

引用次数: 0

Probing Thermal Transitions of Peptide-Major Histocompatibility Complexes by Differential Scanning Fluorimetry. 差示扫描荧光法探测肽-主要组织相容性复合物的热转变。

Methods in molecular biology Pub Date : 2026-01-01 DOI: 10.1007/978-1-0716-5092-9_4

Holly Anne Martin, Lance M Hellman

{"title":"Probing Thermal Transitions of Peptide-Major Histocompatibility Complexes by Differential Scanning Fluorimetry.","authors":"Holly Anne Martin, Lance M Hellman","doi":"10.1007/978-1-0716-5092-9_4","DOIUrl":"https://doi.org/10.1007/978-1-0716-5092-9_4","url":null,"abstract":"Differential scanning fluorimetry (DSF) is a versatile and accessible technique for probing the thermal stability of peptide-major histocompatibility complexes (pMHCs). Understanding the thermal transition midpoint (Tm) between the folded and unfolded states of pMHCs provides critical insights into their structural stability, which is essential for studying antigen presentation and immune responses. pMHC stability is influenced by the inherent structural differences between MHC class I and class II molecules. Notably, class I MHCs require bound peptides for stability, while class II MHCs remain stable without them. Class I MHCs typically present peptides of 8-12 amino acids anchored at specific residues, whereas class II MHCs accommodate longer peptides with distinct anchoring positions. This manuscript outlines a comprehensive DSF protocol optimized for pMHCs, highlighting the method's advantages over other thermal stability techniques, such as differential scanning calorimetry (DSC) and circular dichroism (CD). DSF offers a simpler, cost-effective alternative, utilizing minimal sample volume and readily available real-time PCR (qPCR) instrumentation. We detail critical steps for sample preparation, including optimal buffer selection, dye addition, and degassing procedures, along with specific instrument setup guidelines for both qPCR-based systems and the NanoTemper Prometheus. Data analysis strategies using Microsoft Excel and Origin software are also discussed, including normalization, derivative calculation, and Tm determination. By providing a standardized DSF protocol tailored to pMHC analysis, this manuscript aims to support researchers in efficiently measuring thermal stability, thereby facilitating investigations into pMHC dynamics and immune function.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"3007 ","pages":"43-52"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145889789","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}

引用次数: 0

Direct Inoculation of Pathogenic Yeasts into Mouse Lung by Trans-tracheal Instillation. 经气管滴注法直接在小鼠肺内接种病原菌。

Methods in molecular biology Pub Date : 2026-01-01 DOI: 10.1007/978-1-0716-5019-6_3

Patrícia Campi Santos

{"title":"Direct Inoculation of Pathogenic Yeasts into Mouse Lung by Trans-tracheal Instillation.","authors":"Patrícia Campi Santos","doi":"10.1007/978-1-0716-5019-6_3","DOIUrl":"https://doi.org/10.1007/978-1-0716-5019-6_3","url":null,"abstract":"Pathogenic dimorphic fungi are endemic in certain regions and can cause from subclinical infections to systemic mycoses. These pathogens are associated with high mortality and morbidity rates and represent emerging infectious threats to human populations worldwide. Because most fungal pathogens cause lung infection or use the lung as a route to disseminate to other organs, mammalian pulmonary infection models are crucial tools in the advancement of medical mycology. Many different types of animal models of fungal infection have been developed, with murine models being hailed as the gold standard for studies of pathogenesis, to compare virulence between isolates, preclinical evaluation of vaccines and therapies, and host antifungal immune responses. The ability to control numerous variables in performing the model allows for mimicking human disease states and quantitatively monitoring the course of the disease. To model lung inflammation and injury, fungal infectious propagules can be inoculated via intranasal delivery, intratracheal, or aerosolization approaches. The protocol in this chapter details a method for intratracheal delivery of a fungal suspension, where fungal cells are administered directly into the lungs to initiate infection. The aim is to provide a comprehensive description of techniques required to perform mouse pulmonary infection, such that reproducible results are attained, allowing for the use of this in vivo approach for high-quality studies.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2993 ","pages":"29-42"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145889847","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}

引用次数: 0