Bio-protocol最新文献

{"title":"Rapid and Multiplex Diagnosis of Malaria Using Chelex-100 Extraction and LAMP-MS Assay.","authors":"Min Sup Lim, Young Lan Choe, Woong Sik Jang","doi":"10.21769/BioProtoc.5375","DOIUrl":"10.21769/BioProtoc.5375","url":null,"abstract":"<p><p>Malaria remains a major public health threat, especially in tropical and subtropical regions. Accurate and rapid diagnosis is essential for effective disease management and control, yet conventional malaria diagnostics, including blood smear microscopy using Giemsa staining, PCR, and rapid diagnostic tests (RDTs), are limited by the need for trained personnel, reliance on laboratory infrastructure, and reduced sensitivity at low parasite densities, respectively. This protocol details an innovative, rapid, and economical diagnostic platform combining a simplified Chelex-100 resin-based nucleic acid extraction method with a multiplex loop-mediated isothermal amplification microscanner (LAMP-MS) assay. The malaria diagnostic platform enables simultaneous detection of <i>Plasmodium falciparum</i> (Pf), <i>Plasmodium vivax</i> (Pv), pan-malaria (Pan), and an internal control (IC) within 40 min, from DNA extraction to result interpretation. It demonstrates sensitivity and specificity comparable to traditional PCR-based diagnostics, making it a practical and scalable solution for use in resource-constrained environments. Key features • Low-cost and simple DNA extraction using Chelex-100 resin. • Multiplex detection of Pan malaria, Pf, Pv, and IC using microchip chambers. • Visual endpoint detection using a microscope or a microscanner. • Field-deployable with minimal equipment.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 13","pages":"e5375"},"PeriodicalIF":1.0,"publicationDate":"2025-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12245633/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628095","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}

{"title":"Quantification of Neural Progenitor Cells From Zika Virus-Infected Zebrafish Embryos.","authors":"Yago C P Gomes, Aïssatou Aïcha Sow, Shunmoogum A Patten, Laurent Chatel-Chaix","doi":"10.21769/BioProtoc.5377","DOIUrl":"10.21769/BioProtoc.5377","url":null,"abstract":"<p><p>Zika virus (ZIKV), an arthropod-borne orthoflavivirus, has emerged as a global health concern due to its ability to cause severe fetal neurological disorders, leading to the congenital Zika syndrome (CZS) in neonates. Vertical transmission during pregnancy can alter neural progenitor cell (NPC) proliferation and differentiation and induce apoptosis, leading to microcephaly and other neurodevelopmental abnormalities. While mammalian models have been used to study the impact of ZIKV on NPC behavior, limitations such as high costs, dedicated time, and ethical constraints have fostered the exploration of alternative systems. The zebrafish embryo constitutes an advantageous in vivo model for studying ZIKV neuropathogenesis. Indeed, ZIKV infection phenocopies several features of the CZS while sharing a conserved neuroanatomical layout and offering genetic plasticity and unique accessibility to the infected brain compared to mammals. Here, we describe a protocol for characterizing ZIKV-induced defects of NPCs in this zebrafish model, relying on whole animal flow cytometry. Key features • This protocol introduces a novel and straightforward zebrafish-based system for studying ZIKV-induced neuropathogenesis. • The protocol employs an unbiased methodology for flow cytometry-based quantification of neural progenitor cells from whole embryos. • This protocol can be transposed to any transgenic reporter zebrafish lines producing specific fluorescent cell populations.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 13","pages":"e5377"},"PeriodicalIF":1.0,"publicationDate":"2025-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12245632/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628093","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}

{"title":"Two-photon (2P) Microscopy to Study Ca²⁺ Signaling in Astrocytes From Acute Brain Slices.","authors":"Annamaria Lia, Micaela Zonta","doi":"10.21769/BioProtoc.5371","DOIUrl":"10.21769/BioProtoc.5371","url":null,"abstract":"<p><p>Since the discovery that astrocytes are characterized by Ca²⁺-based excitability, investigating the function of these glial cells within the brain requires Ca²⁺ imaging approaches. The technical evolution from chemical fluorescent Ca²⁺ probes with low cellular specificity to genetically encoded indicators (GECIs) has enabled detailed analysis of the spatial and temporal features of intracellular Ca²⁺ signal. Different imaging methodologies allow the extraction of distinct information on calcium signals in astrocytes from brain slices, with resolution ranging from cell populations to single cells up to subcellular domains. • Here, we describe 2-photon laser scanning microscopy (2PLSM) Ca²⁺ imaging in astrocytes from the somatosensory cortex (SSCx) of adult mice in ex vivo acute cortical slices, performed using two genetically encoded Ca²⁺ indicators, i.e., cytosolic GCaMP6f and endoplasmic reticulum-targeted G-CEPIA1<i>er</i>. The main advantage of the 2PLSM technique, compared to single-photon microscopy, is the possibility to go deeper in the tissue while avoiding photodamage, by limiting laser excitation to a single focal plane. The fluorescent signal of the indicator is analyzed offline in different compartments-soma, proximal processes, and microdomains-for GCaMP6f experiments and in the perinuclear, somatic area for G-CEPIA1<i>er</i>. The analysis of Ca²⁺ signal from different compartments, although not providing a value of absolute concentration, allows a critical comparison of the degree of astrocyte activation between different experimental conditions or mouse models. Moreover, the analysis of G-CEPIA1<i>er</i> signal, which reveals metabotropic receptor activation as a dynamic decrease in free Ca²⁺ in the endoplasmic reticulum (ER), can provide information on possible alterations in this critical second messenger pathway in astrocytes, including, for example, steady-state ER Ca²⁺ levels and kinetics of Ca²⁺ release. Key features • This protocol is useful to characterize basal and evoked Ca²⁺ astrocyte activity in acute mouse brain slices, deepening analysis to different subcellular territories and compartments. • The induction of Ca²⁺ probe expression requires surgical experience in mice and appropriate stereotaxic equipment for adeno-associated viral (AAV) vector injection. • The imaging experimental protocol takes approximately 8 h from the beginning of brain slice preparation to completion of 2PLSM imaging. • The described protocol, from slice preparation to signal analysis, can also be adapted for astrocyte Ca²⁺ experiments using epifluorescence or confocal microscopy.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 13","pages":"e5371"},"PeriodicalIF":1.0,"publicationDate":"2025-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12245626/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628096","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}

{"title":"Visualization of the Evolution and Transmission of Circulating Vaccine-Derived Poliovirus (cVDPV) Outbreaks in the African Region.","authors":"Collins D Owuor, Brook Tesfaye, Arthur Yannick Doungmo Wakem, Sakma Kabore, Caroline Obianuju Ikeonu, Mariette Egbonoumi Fifatin Glitho Epse Doussoh, Priscilla Epse Mosoke Bobimwoh Sigala, Idris Ibrahim Ibrahim, Abdullateef Jimoh, Idah Ndumba, Jermaine Khumalo, David O Oviaesu, Cheroigin Kipchirchir, Carolyne Gathenji, John Kipterer, Kebba Touray, Hamisu Abdullahi, Kathleen Rankin, Ousmane M Diop, Julius E Chia, Ndoutabe Modjirom, Jamal A Ahmed, Anfumbom K W Kfutwah","doi":"10.21769/BioProtoc.5376","DOIUrl":"10.21769/BioProtoc.5376","url":null,"abstract":"<p><p>Since the creation of the Global Polio Eradication Initiative (GPEI) in 1988, significant progress has been made toward attaining a poliovirus-free world. This has resulted in the eradication of wild poliovirus (WPV) serotypes two (WPV2) and three (WPV3) and limited transmission of serotype one (WPV1) in Pakistan and Afghanistan. However, the increased emergence of circulating vaccine-derived poliovirus (cVDPV) and the continued circulation of WPV1, although limited to two countries, pose a continuous threat of international spread of poliovirus. These challenges highlight the need to further strengthen surveillance and outbreak responses, particularly in the African Region (AFRO). Phylogeographic visualization tools may provide insights into changes in poliovirus epidemiology, which can in turn guide the implementation of more strategic and effective supplementary immunization activities and improved outbreak response and surveillance. We created a comprehensive protocol for the phylogeographic analysis of polioviruses using Nextstrain, a powerful open-source tool for real-time interactive visualization of virus sequencing data. It is expected that this protocol will support poliovirus elimination strategies in AFRO and contribute significantly to global eradication strategies. These tools have been utilized for other pathogens of public health importance, for example, SARS-CoV-2, human influenza, Ebola, and Mpox, among others, through real-time tracking of pathogen evolution (https://nextstrain.org), harnessing the scientific and public health potential of pathogen genome data. Key features • Employs Nextstrain (https://nextstrain.org), which is an open-source tool for real-time interactive visualization of genome sequencing datasets. • First comprehensive protocol for the phylogeographic analysis of poliovirus sequences collected from countries in the World Health Organization (WHO) African Region (AFRO). • Phylogeographic visualization may provide insights into changes in poliovirus epidemiology, which can in turn guide the implementation of more strategic and effective vaccination campaigns. • This protocol can be deployed locally on a personal computer or on a Microsoft Azure cloud server for high throughput.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 13","pages":"e5376"},"PeriodicalIF":1.0,"publicationDate":"2025-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12245628/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628098","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}

{"title":"Evaluation of In Vitro Cytotoxic Activity of CAR-T Cells Using Patient-Derived Organoids.","authors":"Lei Yu","doi":"10.21769/BioProtoc.5369","DOIUrl":"10.21769/BioProtoc.5369","url":null,"abstract":"<p><p>Adoptive immune cell therapy, especially chimeric antigen receptor T (CAR-T) cells, has emerged as a promising strategy in solid tumor treatment, owing to its unique ability to specifically recognize and effectively eliminate tumor cells. Patient-derived organoids (PDOs) offer a robust and physiologically relevant platform for assessing the safety and efficacy of CAR-T-cell-based therapies. We now describe a detailed protocol for an in vitro evaluation system based on the co-culture of PDOs and CAR-T cells. This system encompasses the establishment of tumor organoids from patient tumor specimens, the isolation of T cells from matched peripheral blood mononuclear cells (PBMCs), and the generation of antigen-specific CAR-T cells. Through the use of fluorescent labeling to visualize different cells and apoptosis-related events post-interaction, along with quantitative analyses of T-cell proliferation, tumor organoid apoptosis, and the secretion of immune effector molecules, this system enables a robust and multifaceted evaluation of CAR-T cell cytotoxicity in vitro. Collectively, this co-culture system provides a systematic and reproducible in vitro platform for evaluating the functional activity of CAR-T cells and advancing research in tumor immunology and immunotherapy.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 13","pages":"e5369"},"PeriodicalIF":1.0,"publicationDate":"2025-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12245622/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628087","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}

{"title":"Construction of Large Cranial Windows With Nanosheet and Light-Curable Resin for Long-term Two-Photon Imaging in Mice.","authors":"Taiga Takahashi, Yu Makino, Yosuke Okamura, Tomomi Nemoto","doi":"10.21769/BioProtoc.5373","DOIUrl":"10.21769/BioProtoc.5373","url":null,"abstract":"<p><p>In vivo two-photon imaging of the mouse brain is essential for understanding brain function in relation to neural structure; however, its application is limited by the size and mechanical stability of conventional cranial windows. Here, we present the procedure of a large-scale cranial window technique based on the nanosheet incorporated into light-curable resin (NIRE) method. This approach utilizes a biocompatible polyethylene-oxide-coated CYTOP (PEO-CYTOP) nanosheet combined with light-curable resin, allowing the window to conform to the brain's curved surface. The protocol enables long-term, high-resolution, and multiscale imaging-from subcellular structures to large neuronal populations-in awake mice over several months. Key features • This protocol establishes large-scale cranial windows by combining a flexible, biocompatible PEO-CYTOP nanosheet with light-curable resin. • The large-scale cranial window provides long-term optical transparency and mechanical stability, enabling chronic in vivo imaging with minimal motion artifacts. • This approach facilitates multiscale two-photon imaging-from subcellular structures to large-scale neural networks-in awake mice.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 13","pages":"e5373"},"PeriodicalIF":1.0,"publicationDate":"2025-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12242551/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628085","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}

{"title":"Kinase Mobility Shift Assay (KiMSA) for Assessing Protein Kinase A Activity.","authors":"Analia G Novero, Tomás J Steeman, Catalina Curcio, Lara Buccolini, Andres Binolfi, Diego Krapf, Mariano G Buffone, Dario Krapf, Cintia Stival","doi":"10.21769/BioProtoc.5366","DOIUrl":"10.21769/BioProtoc.5366","url":null,"abstract":"<p><p>The cAMP-dependent protein kinase (PKA) is one of the most extensively distributed kinases among intracellular signal cascades, with a pivotal role in the regulation of various processes, including the capacitation of sperm cells. Traditional assessments of PKA activity rely on the utilization of [γ-³²P] ATP and the Kemptide peptide as a substrate. This strategy presents several major drawbacks, including high costs and health risks derived from the manipulation of radioactive isotopes. In this work, we introduce an enhanced non-radioactive assay to quantify PKA activity, termed kinase mobility shift assay (KiMSA), based on the use of a fluorescent-labeled Kemptide (Kemptide-FITC). Once the kinase reaction is terminated, the products can be easily resolved through electrophoresis on an agarose gel and quantified by fluorescence densitometry. We show that KiMSA is suitable for isolated PKA as well as for the enzyme in cell extracts. In addition, it enables quantification of PKA activity during the progression of mouse sperm capacitation. Furthermore, the assay enables monitoring the inhibition of PKA with pharmacological inhibitors in live cells. Therefore, the experimental and optimal assay conditions are set so that KiMSA can be used to assess in vitro as well as in vivo PKA activity in sperm cells. Finally, this method allows for measurement of cAMP concentrations, rendering a versatile technique for the study of cAMP/PKA pathways. Key features • KiMSA is a versatile kinase mobility shift assay for measuring PKA activity in sperm physiology, replacing radioactive assays with a fluorescence-labeled substrate for high sensitivity. • This in vitro assay enables evaluation of activation state, drug effects, or PKA kinetics after purification or mutation. • This assay measures PKA activity from cell extracts, reflecting pre-lysis activation status and intracellular signaling, though not a true in vivo readout. • The standard protocol completion time is one day, including sperm preparation, kinase reactions, electrophoresis, and fluorescence quantification.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 13","pages":"e5366"},"PeriodicalIF":1.0,"publicationDate":"2025-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12245630/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628090","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}

{"title":"In Vitro Co-culture of Bacterial and Mammalian Cells to Investigate Effects of Potential Probiotics on Intestinal Barrier Function.","authors":"Ajitpal Purba, Rachel Claire Anderson, Dulantha Ulluwishewa","doi":"10.21769/BioProtoc.5361","DOIUrl":"10.21769/BioProtoc.5361","url":null,"abstract":"<p><p>Human intestinal barrier function is crucial for health. Beneficial microbes, such as commensal gut bacteria and probiotics, are known to contribute to the regulation of this barrier function. Interactions between bacteria and human intestinal cells can be analyzed by co-culturing bacteria with mammalian cells in vitro. Here, we describe a method to assess the effect of individual bacterial strains on intestinal barrier function using automated transepithelial electrical resistance (TEER) measurements. Caco-2 cells are used as a model of the intestinal epithelium, as these cells spontaneously differentiate into small intestinal epithelial-like cells characterized by tight junctions between adjacent cells. These cells are seeded on polyester filter inserts and cultured for 17 days to form a differentiated monolayer prior to the co-culture experiment. Bacteria are grown on agar, and a single colony is used to prepare a liquid culture in bacterial broth appropriate for the bacteria of interest. On the day of the co-culture experiment, the bacterial culture is resuspended in cell culture medium at the desired concentration. Inserts are transferred to cellZscope cell modules to enable automated TEER measurements, and the medium in the insert is replaced with cell culture medium containing the bacteria of interest. This method allows for intestinal tight junction barrier function to be assessed non-invasively and in real-time in response to probiotics. The use of the automated cellZscope system eliminates the need for labor-intensive manual TEER measurements, which reduces the variability in data that results from human handling and temperature changes that occur when cells are removed from the incubator. Key features • Real-time measurement of the effects of aerobic or aerotolerant-anaerobic probiotics on intestinal barrier integrity. • Automated TEER measurements are achieved by a nanoAnalytics CellZscope instrument. • Requires at least four weeks to complete one experimental run, including the revival and growth of mammalian cells in flasks, prior to differentiation in inserts. • Completing the recommended three experimental runs, staggered by one week, takes six weeks.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 12","pages":"e5361"},"PeriodicalIF":1.0,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12229796/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144585779","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}

{"title":"Isolation and Characterization of Cervical Cancer-Associated Mesenchymal Stem Cells From Primary Tumors Using Explant Culture.","authors":"Surbhi Singla, Sanchita Khurana, Rashmi Bagga, Radhika Srinivasan, Shalmoli Bhattacharyya","doi":"10.21769/BioProtoc.5358","DOIUrl":"10.21769/BioProtoc.5358","url":null,"abstract":"<p><p>Cancer-associated mesenchymal stem cells (Ca-MSCs), an integral part of the tumor microenvironment, play a major role in modulating tumor progression; they have been reported to progress as well as inhibit various cancers, including cervical cancer. To understand the exact role of Ca-MSCs in tumor modulation, it is necessary to have an optimized protocol for Ca-MSCs isolation. This work demonstrates the isolation and expansion of a primary culture of cervical cancer-associated MSCs (CCa-MSCs) from the biopsy sample of cervical cancer patients using the explant culture technique. The isolated cells were characterized according to International Society for Cellular Therapy (ISCT) guidelines. Morphological analysis revealed that cells were adherent to the plastic surface and possessed spindle-shaped morphology. Flow cytometry analysis of the cells showed high expression (~98%) for MSC-specific cell surface markers (CD90, CD73, and CD105), negative expression (<0.5%) for endothelial cell marker (CD34) and hematopoietic cell marker (CD45), and negligible expression for HLA-DR, as recommended by ISCT. Further, trilineage differentiation potential analysis of the cells showed their osteogenic and chondrogenic potential and adipogenic differentiation. This standardized protocol will assist in the cultivation of CCa-MSCs and the study of their interactions with tumor cells and other components of the tumor microenvironment. This protocol may be utilized in the establishment of Ca-MSCs from other types of cancers as well. Key features • Isolation and expansion of cervical cancer-associated mesenchymal stem cells (CCa-MSCs) from patient biopsy sample. • Characterization of isolated CCa-MSCs for the presence of MSC-specific cell surface markers and trilineage differentiation potential. • CCa-MSCs can be employed to study the interactions with the tumor cells in the tumor microenvironment. Graphical overview.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 12","pages":"e5358"},"PeriodicalIF":1.0,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12222631/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144577229","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}

{"title":"An Improved m6A-ELISA for Quantifying <i>N⁶</i> -methyladenosine in Poly(A)-purified mRNAs.","authors":"Wei Yee Chan, Waleed S Albihlal, Folkert J Van Werven","doi":"10.21769/BioProtoc.5359","DOIUrl":"10.21769/BioProtoc.5359","url":null,"abstract":"<p><p><i>N⁶</i> -methyladenosine (m6A) is an abundant internal mRNA modification with roles in regulating cellular and organismal physiology, including development, differentiation, and disease. The deposition of m6A is highly regulated, with various m6A levels across different environmental conditions, cellular states, and cell types. Available methods for measuring bulk m6A levels are often time-consuming, have low throughput, and/or require specialized instrumentation or data analyses. Here, we present a detailed protocol for measuring bulk m6A levels in purified poly(A) RNA samples with m6A-ELISA using a standard-based approach. Critical steps of the protocol are highlighted and optimized, including poly(A) RNA quality controls and antibody specificity testing. The protocol is fast, scalable, adaptable, and cost-effective. It does not require specialized instrumentation, training, or skills in data analysis. We have successfully tested this protocol on mRNAs isolated from budding yeast and mouse cell lines. Key features • <i>N⁶</i> -methyladenosine quantification, including mRNA isolation, can be achieved in two days. • Describes a robust method for poly(A) RNA isolation from total RNA, minimizing non-poly(A) RNA contamination. • Based on Ensinck et al. [1], optimizing poly(A) RNA selection from yeast and mammalian cells and reducing the amount of mammalian mRNA for the assay.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 12","pages":"e5359"},"PeriodicalIF":1.0,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12222634/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144577225","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}