Bio-protocol最新文献

{"title":"The Centriole Stability Assay: A Method to Investigate Mechanisms Involved in the Maintenance of the Centrosome Structure in <i>Drosophila</i> Cultured Cells.","authors":"Mariana Lince-Faria, Ana Ferreira-Silva, Ana Pimenta-Marques","doi":"10.21769/BioProtoc.5330","DOIUrl":"10.21769/BioProtoc.5330","url":null,"abstract":"<p><p>Centrosomes are vital eukaryotic organelles involved in regulating cell adhesion, polarity, mobility, and microtubule (MT) spindle assembly during mitosis. Composed of two centrioles surrounded by the pericentriolar material (PCM), centrosomes serve as the primary microtubule-organizing centers (MTOCs) in proliferating cells. The PCM is crucial for MT nucleation and centriole biogenesis. Centrosome numbers are tightly regulated, typically duplicating once per cell cycle, during the S phase. Deregulation of centrosome components can lead to severe diseases. While traditionally viewed as stable structures, centrosomes can be inactivated or disappear in differentiating cells, such as epithelial cells, muscle cells, neurons, and oocytes. Despite advances in understanding centrosome biogenesis and function, the mechanisms maintaining mature centrosomes or centrioles, as well as the pathways regulating their inactivation or elimination, remain less explored. Studying centrosome maintenance is challenging as it requires the uncoupling of centrosome biogenesis from maintenance. Tools for acute spatial-temporal manipulation are often unavailable, and manipulating multiple components in vivo is complex and time-consuming. This study presents a protocol that decouples centrosome biogenesis from maintenance, allowing the study of critical factors and pathways involved in the maintenance of the integrity of these important cellular structures. Key features • <i>Drosophila</i> cultured cells are resistant to centriole reduplication during S phase arrest, making them a suitable model for studying centrosome integrity without confounding effects from centriole biogenesis.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 11","pages":"e5330"},"PeriodicalIF":1.0,"publicationDate":"2025-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12152112/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144287567","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":"Murine Osteoblast and Osteoclast Co-culture on Demineralized Bone Paper for Bone Remodeling.","authors":"Seema Amin, Hyejin Yoon, Dong-Hee Choi, Yi-Hao Hsu, Jungwoo Lee","doi":"10.21769/BioProtoc.5339","DOIUrl":"10.21769/BioProtoc.5339","url":null,"abstract":"<p><p>Continuous and balanced bone remodeling is essential for maintaining mechanical integrity, mineral homeostasis, and hematopoiesis. Dysregulated bone metabolism develops pathological conditions, such as osteoporosis and bone metastasis. Functional and analytical recapitulation of bone remodeling in vitro is critical for advancing our understanding of bone mineral metabolism, disease mechanisms, and drug development. However, conventional models fail to replicate the essential complexity of the bone extracellular matrix (ECM) and the dynamic interplay between bone-forming osteoblasts and bone-resorbing osteoclasts. Recently, we developed an osteoid-mimicking demineralized bone paper (DBP) by thin-sectioning demineralized bovine compact bone matrix. DBP supports osteoblastic mineral deposition and the subsequent transition to bone-lining cells. When co-cultured with bone marrow mononuclear cells under biochemical stimulation, osteoblasts shift their regulatory secretion profiles and effectively induce osteoclastogenesis. The semi-transparent nature of DBP, combined with primary osteogenic cells retrieved from DsRed and eGFP reporter mice, enables longitudinal fluorescent monitoring of these multicellular processes and quantitative analysis. In this protocol, we describe the methods for DBP generation, reconstituting mineralized bone tissue complexity with osteoblasts, and recapitulating the bone remodeling cycle through bone marrow monocytes co-culture under biochemical stimulation, offering a useful platform for the related and broader research community. Key features • DBP supports in vivo-relevant osteoblastic mineral deposition and transition to lining cells. • DBP supports in vivo-relevant recapitulation of osteoblast-osteoclast-driven bone remodeling. • DBP supports longitudinal fluorescent monitoring of multicellular processes and quantification.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 11","pages":"e5339"},"PeriodicalIF":1.0,"publicationDate":"2025-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12152114/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144287550","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 Automated Imaging Method for Quantification of Changes to the Endomembrane System in Mammalian Spheroid Models.","authors":"Margaritha M Mysior, Jeremy C Simpson","doi":"10.21769/BioProtoc.5331","DOIUrl":"10.21769/BioProtoc.5331","url":null,"abstract":"<p><p>Three-dimensional cell models, such as spheroids, represent a more physiological arrangement in which cells can grow, allowing them to develop cell-cell interactions in all dimensions. The most common methods for growing spheroids are scaffold-based, typically using either extracellular matrix or hydrogels as a physical support for the cellular assembly. One key problem with this approach is that the spheroids that are produced can be highly variable in size and shape. The protocol presented here allows for the systematic production of uniform spheroids in a short time frame by utilising a micropatterned plate. We show that spheroids can be used to investigate fundamental research questions, such as how the endomembrane system is organised in cells. Our protocol can be used in a manual or automated manner, potentially allowing scaling up for screening applications. Furthermore, without the complication of removing the spheroids from the extracellular matrix or hydrogel, as would be required in scaffold-based systems, spheroids can easily be used in other downstream applications. Key features • This work builds upon the method developed by Monjaret et al. [1] and establishes a robust method to produce spheroids from HeLa Kyoto cells. • This protocol generates consistent populations of spheroids that can be used to investigate organelle biology and membrane trafficking pathways. • Entire spheroids can be analysed in a volumetric manner. • This method can be used in both manual and automated pipelines, thereby facilitating use in high-throughput and high-content screens.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 11","pages":"e5331"},"PeriodicalIF":1.0,"publicationDate":"2025-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12152110/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144287521","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 Condensation Assay of Fluorescent Protein-Fused PRPP Amidotransferase Purified from Budding Yeast Cells.","authors":"Masak Takaine","doi":"10.21769/BioProtoc.5335","DOIUrl":"10.21769/BioProtoc.5335","url":null,"abstract":"<p><p>De novo synthesis of purine nucleotide is essential for the production of genetic materials and cellular chemical energy. PRPP amidotransferase (PPAT) is the rate-limiting enzyme in de novo purine synthesis, thereby playing a crucial regulatory role in this pathway. Recent studies suggest that metabolic enzymes, including PPAT, form condensates through phase separation to regulate cellular metabolism in response to environmental changes. However, due to the lack of methods for purifying eukaryotic PPAT, the biophysical properties of the enzyme have remained unknown. Here, I describe a protocol for purifying budding yeast PPAT tagged with green fluorescent protein from yeast cells, as well as an in vitro assay to examine condensation of the fluorescent PPAT by microscopy. These techniques enabled us to elucidate the mechanism controlling PPAT condensation and may also be applicable to the purification and condensation assay of other enzymes. Key features • First example of purification of fluorescent protein-fused PPAT. • Time-saving, one-step affinity purification without expensive equipment. • Automated image and data processing increases throughput and reproducibility and ensures reliability in the study of biomolecular condensates. • A powerful alternative for protein purification to bacterial expression systems.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 11","pages":"e5335"},"PeriodicalIF":1.0,"publicationDate":"2025-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12152107/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144287546","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":"High-Throughput Screening Identification of Chemical Compounds That Affect Cold-Regulated Gene Expression in <i>Arabidopsis thaliana</i> Using an Excised Single Leaf.","authors":"Kohei Kitawaki, Ryota Mihara, Yausko Ito-Inaba, Takehito Inaba","doi":"10.21769/BioProtoc.5319","DOIUrl":"10.21769/BioProtoc.5319","url":null,"abstract":"<p><p>The identification of chemical compounds that affect intracellular processes has greatly contributed to the understanding of developmental regulation in plants. In this protocol, we describe a method for identifying chemical compounds that affect cold-regulated gene expression in <i>Arabidopsis thaliana</i>. Specifically, we generated <i>Arabidopsis</i> plants harboring a COLD-REGULATED 15A <i>(COR15A)</i> promoter::luciferase (<i>COR15Apro::LUC</i>) construct and grew them in a submerged liquid culture. Using a single true leaf excised from <i>COR15Apro::LUC</i> plants and 96-well culture plates, we performed high-throughput screening of chemical compounds that inhibit cold-induction of <i>COR15Apro::LUC</i>. Luciferase activity was detected using a microplate reader and a chemiluminescence imaging device. This protocol can be easily used for the identification of chemical compounds that regulate other processes, being versatile with respect to equipment. Key features • High-throughput screening of chemical compounds that affect cold-regulated gene expression is possible using a single leaf excised from <i>Arabidopsis</i> grown in a submerged culture. • Screening is based on luciferase activity derived from an excised single leaf. • Direct measurement of luciferase activity is possible using a microplate reader and a chemiluminescence imaging device. • This protocol can be easily used for the identification of chemical compounds that regulate other processes.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5319"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104835/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164098","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":"I-PREFR: Inverse PCR-Based Restriction Enzyme FRee Unidirectional Strategy for Rapid Markerless Chromosomal Gene Deletion and Reconstitution in Bacteria Using Suicide Vectors.","authors":"Rekha Rana, Anushika Sharma, Ashish Dutta, Prabhu B Patil","doi":"10.21769/BioProtoc.5314","DOIUrl":"10.21769/BioProtoc.5314","url":null,"abstract":"<p><p>The standard protocols for allelic exchange using homologous recombination deploy suicide vectors with negative selection markers. However, the use of multiple restriction enzymes to generate sticky ends in the vector and the insert for cloning is time-consuming, resource-intensive, and challenging. The advent of next-generation proofreading enzymes is enabling researchers to routinely carry out long-range PCR. Hence, amplifying 5-6 kb of complete low-complex DNA cloning vectors and 2-3 kb of complex genomic regions is much easier. Here, we report a simple, accurate, rapid, and unidirectional approach for chromosomal in-frame gene deletion and complementation by reconstitution of the full-length gene without using any restriction enzymes. The method requires long-range PCR using Phusion polymerase to linearize the vector and amplify the target gene to create a recombinant vector (pRM1) and further inverse PCR amplification of pRM1 to create a recombinant vector (pRM4) with a deleted version of the gene. The cloning steps involve the use of kinase and ligase for phosphorylation and ligation steps, respectively. The recombinant plasmid, pRM4, is finally transformed into electrocompetent cells of <i>Xanthomonas sontii</i>, a gram-negative phytobacterium, for final genomic integration/excision to obtain an in-frame gene deletion mutant (PPL1RM15). Gene reconstitution for complementation is carried out by electroporating the deletion mutant with the recombinant plasmid (pRM1) carrying the wild-type allele. Clean gene mutation, allele restoration, and plasmid excision are confirmed using whole-genome sequencing. Key features • The protocol is cost-effective and simple, eliminating the need for restriction enzymes and multiple sets of lengthy primers with restriction sites. • The protocol requires only kinase, ligase, and polymerase, along with three sets of standard-sized desalted primers. • The protocol is unidirectional; no need to create two different recombinant plasmids for gene deletion and complementation by reconstitution of the full-length gene. • The difference in size between empty and recombinant vectors facilitates the easy screening of transformed <i>Escherichia coli</i> colonies through colony PCR. • The strategy can be applied to any bacteria using a suitable suicide vector with appropriate positive and negative selection markers.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5314"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104833/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164101","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":"Development of Polyethylene Glycol Diacrylate-Based Micropattern Substrate to Study the Interplay Between Surface Topography and Cellular Response for Tissue Engineering Applications.","authors":"Mohd Khairul Akma Darwis, Victoria Levario-Diaz, Sadaf Pashapour, Jonah Luka Voigt, Eloïse Lebaudy, Norhayati Sabani, Ahmad Shuhaimi Abu Bakar, Nihal Engin Vrana, Philippe Lavalle, Elisabetta Ada Cavalcanti-Adam, Siti Hawa Ngalim","doi":"10.21769/BioProtoc.5323","DOIUrl":"10.21769/BioProtoc.5323","url":null,"abstract":"<p><p>A key goal in the bioengineering field is the development of surface patterning of proteins that guide and control cellular organization. To this end, we developed a method to create a microstructured hydrogel based on soft-lithography techniques using polydimethylsiloxane (PDMS) and polyethylene glycol diacrylate (PEGDA). This approach involves the design of microfluidic geometries using graphical software, employing PDMS as a mold and leaving PEGDA as a substrate for the fabrication of microstructures and, thus, patterning extracellular matrix (ECM) proteins to promote cell adhesion. The combination of these techniques allows the fabrication of hydrogel microstructures without following conventional photolithography methods, such as the use of a photomask, the alignment required to produce the patterns, and the associated expenses. This study highlights the versatility and potential of PEGDA-based hydrogels as platforms to advance tissue engineering strategies. Key features • This protocol focuses on investigating the feasibility of patterning PEGDA as a substrate for protein surface patterning and further tissue engineering applications. • Optimization of the fabrication of PEGDA hydrogels into simple shapes and angular patterns, ensuring a robust substrate capable of guiding cellular responses.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5323"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104878/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164039","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":"Quantifying Thrombogenicity: A Bioanalytical Protocol for the Absorbance-Based Assessment of Vascular Implants with Plasma.","authors":"Anna Sallee, Aurora Battistella, Richard Johnson, Alexander Duong, Wei Tan, Novella M Keeling","doi":"10.21769/BioProtoc.5316","DOIUrl":"10.21769/BioProtoc.5316","url":null,"abstract":"<p><p>Assessing thrombogenicity is crucial for evaluating biomaterials, especially those that interface with flowing blood, such as cardiovascular implants, including vascular stents, grafts, and stent-grafts. To standardize thrombogenicity assessments, we use human plasma and quantify the light absorbance associated with the biomaterial. For this evaluation, various tubular vascular implants from leading brands-such as bare-metal stents, drug-eluting stents, vascular grafts, and stent-grafts-are longitudinally sectioned into small pieces and placed in a low-adhesion 96-well plate. Using either platelet-rich plasma (PRP) or platelet-poor plasma (PPP), we measure the absorbance of light passing through the plate over an hour and plot the resulting curve. This method quantifies the thrombogenicity of a biomaterial under controlled conditions. Key factors examined include anticoagulants, platelet presence, and surface-coating molecules to assess their impact on thrombogenicity. Using this simple, reproducible protocol, we demonstrated (a) the relative efficacy of various anticoagulants in thrombogenicity assessments, and (b) the effectiveness of vascular coating molecules in reducing thrombogenicity on stents. This streamlined approach offers valuable insights for optimizing biomaterial performance in vascular implants. Unlike conventional clotting assays, which focus on standardized blood clotting mechanisms, this assay is tailored to evaluate biomaterials and external parameters influencing thrombogenicity. Key features • This protocol is a static, in vitro, quantitative assessment of thrombogenicity for both novel and commercial vascular biomaterials with flat or tubular geometries. • Thrombogenicity is assessed by evaluating the opaqueness of platelet-rich or platelet-poor plasma over time to measure fibrin formation. • Various anticoagulants such as sodium citrate, sodium ethylenediaminetetraacetic acid, and sodium heparin are compared to evaluate the efficacy of the protocol. • This protocol compares the thrombogenicity of various commercial stents, grafts, and stent-graft hybrids, as well as novel molecular coatings.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5316"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104834/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164049","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":"Stable ¹³C-glutamine Tracing Resolved Metabolomics for Cancer Metabolism Study.","authors":"Yaogang Zhong, Liqing He, Xinmin Yin, Logan Mazik, Xiang Zhang, Deliang Guo","doi":"10.21769/BioProtoc.5322","DOIUrl":"10.21769/BioProtoc.5322","url":null,"abstract":"<p><p>Stable isotopes have frequently been used to study metabolic processes in live cells both in vitro and in vivo. Glutamine, the most abundant amino acid in human blood, plays multiple roles in cellular metabolism by contributing to the production of nucleotides, lipids, glutathione, and other amino acids. It also supports energy production via anaplerosis of tricarboxylic acid cycle intermediates. While ¹³C-glutamine has been extensively employed to study glutamine metabolism in various cell types, detailed analyses of specific lipids derived from ¹³C-glutamine via the reductive carboxylation pathway are limited. In this protocol, we present a detailed procedure to investigate glutamine metabolism in human glioblastoma (GBM) cells by conducting ¹³C-glutamine tracing coupled with untargeted metabolomics analysis using liquid chromatography-mass spectrometry (LC-MS/MS). The method includes step-by-step instructions for the extraction and detection of polar metabolites and long-chain fatty acids (LCFAs) derived from ¹³C-glutamine in GBM cells. Notably, this approach enables the distinction between isomers of two monounsaturated FAs with identical masses: palmitoleic acid (16:1n-7) (cis-9-hexadecenoic acid) and palmitelaidic acid (16:1n-7) (trans-9-hexadecenoic acid) derived from ¹³C-glutamine through the reductive carboxylation process. In addition, using this protocol, we also unveil previously unknown metabolic alterations in GBM cells following lysosome inhibition by the antipsychotic drug pimozide. Key features • Methods for analyzing the flux of the stable isotope ¹³C-glutamine in cancer cells and identifying its derived polar metabolites and long-chain fatty acids (LCFAs). • Distinguishes isomers of long-chain fatty acids, such as palmitoleic acid (16:1n-7) (cis-9-Hexadecenoic acid) and palmitelaidic acid (16:1n-7) (trans-9-Hexadecenoic acid), which share the exact same mass. • The method is utilized to investigate glutamine metabolism reprogramming in cancer cells following lysosome inhibition.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5322"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104877/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164283","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":"RNA PolyA Tailing Assay to Qualitatively Analyze Circular RNA Manufacturing.","authors":"Zih-Shiuan Chuang, Chan-I Su, Hsin-I Wang, Chia-Yi Yu","doi":"10.21769/BioProtoc.5310","DOIUrl":"10.21769/BioProtoc.5310","url":null,"abstract":"<p><p>A covalently closed loop structure provides circular RNA (circRNA) with more stability than conventional RNAs in linear form, making circRNA an emerging tool in RNA therapeutics. The qualification and quantification of circRNA after production is critical for its design and effectiveness assessments, particularly when the following applications could be affected by byproduct RNAs. Despite PCR-based methods effectively detecting low-abundance circRNA, they are unsuitable for assessing uncircularized RNA in a mass production fraction to maintain quality control. Here, we present a straightforward protocol for evaluating uncircularized byproduct RNAs from circRNA production. This method enrolls the template-independent RNA polymerase activity to add adenine tails (polyA) to the 3' ends of a linear RNA, making it easy to distinguish trace byproducts or uncircularized RNA from a pool of mass circRNA products. With conventional linear RNA and RNase R-treated circRNA as the positive and negative controls, the purity of a circRNA preparation could be readily resolved. Regardless of circRNA production strategies, this protocol provides a reliable and practical way to ensure the consistent quality of homemade circRNAs or to recheck circRNA quality from commercial manufacturing. Key features • The RNA circularization assessment relies on detecting a free 3' end OH group of the non-circRNA in the circRNA products. • An RNA denaturing electrophoresis is required to detect nucleotide length changes of the non-circRNA post-3' end tailing. • Rather than quantitatively, the molecular weight changes qualitatively highlight the trace non-circRNA in a circRNA preparation. • While there are different techniques for producing circRNA, the tailing method is effective for most, detecting leftover linear RNA contaminants.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5310"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104830/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164166","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}