Methods and Protocols最新文献

{"title":"A Quick Reverse Transfection and Dual-Imaging Protocol for Simultaneous Viability and Oligonucleotide Delivery Assessment in 4T1 Cells.","authors":"Andrea González-Báez, Raúl Rangel-López, Gustavo Hernández-Vidal, Armando Trejo-Chávez, Luis Rodríguez-Tovar, Diana Zárate-Triviño, Diana Caballero-Hernandez, Rodolfo Nino Fong, Gerardo Méndez-Zamora, Diana Elisa Zamora-Avila","doi":"10.3390/mps9050137","DOIUrl":"10.3390/mps9050137","url":null,"abstract":"<p><p>The 4T1 cell line is a widely used model for triple-negative breast cancer (TNBC) research due to its high metastatic potential and resemblance to clinical progression. However, its biological characteristics often limit the efficiency and reproducibility of conventional nucleic acid delivery protocols. Here, we present an optimized non-viral delivery framework for efficient cell-associated uptake in 4T1 cells. Five carrier systems-Xfect RNA polymer (Xfect), polyethyleneimine (PEI), chitosan (CHI), and gold nanoparticle conjugates (AuPEI and AuCHI)-were systematically evaluated using both standard (adherent) and reverse (suspension) delivery approaches. A fluorescently labeled oligonucleotide (5'-TYE563) served as a reporter cargo at final concentrations of 5 nM and 10 nM, comparing complexation times of 15 s and 15 min. The proposed protocol introduces a rapid complexation strategy combined with reverse delivery, significantly reducing preparation time while enhancing cell-associated uptake. Under optimized conditions (10 nM, 15 s, reverse), Xfect achieved near-complete cellular uptake with minimal cytotoxicity. Crucially, the colocalization of dual-fluorescence signals enabled the simultaneous validation of metabolic viability and internalized cargo within a single field of view, bypassing the need for independent bulk cytotoxicity assays. This methodology provides a rapid, reproducible, and cost-effective framework to standardize early-stage delivery conditions for difficult-to-transfect cell lines such as 4T1.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 5","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13638061/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149030921","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":"Gradient-Free Isolation of Murine Pancreatic Islets for Single-Cell RNA Sequencing.","authors":"Evgeny S Ruchko, Zakhar R Starinnov, Marat S Sabirov, Maria B Chernysheva, Arthur A Lee, Vagif Ali Oglu Gasanov, Andrey V Vasiliev","doi":"10.3390/mps9050136","DOIUrl":"10.3390/mps9050136","url":null,"abstract":"<p><p>Isolation of pancreatic islets for single-cell RNA sequencing (scRNA-seq) remains technically challenging because enzymatic digestion, mechanical dissociation, and subsequent purification can cause cell loss, aggregation, and processing-induced transcriptional changes. This study aimed to adapt a practical gradient-free alternative for preparing murine pancreatic islets for downstream single-cell transcriptomic analysis while reducing the number of sample-processing steps. The workflow combined intraductal perfusion of the pancreas with 3 mL of a collagenase solution through the common bile duct, controlled enzymatic digestion at 37 °C, performed manual islet selection without density-gradient purification, and achieved gentle dissociation with Accutase and EDTA. DNase I was included in all post-digestion steps to limit cell aggregation. The protocol was evaluated using pancreatic islets from BKS.Cg-Dock7^m +/+ Lepr^db/J mice, a model of type 2 diabetes. Compared with Ficoll-based purification, the gradient-free protocol provided viable cell yields within a similar range while eliminating density-gradient preparation, an 18 min gradient centrifugation step, two subsequent wash centrifugations, and the associated transfer and dilution steps. The resulting scRNA-seq library showed satisfactory quality-control (QC) characteristics and retained the major endocrine populations, including β-, α-, δ-, and PP cells. The gradient-free sample also showed a lower overall stress-response score and lower expression of several immediate early genes, although heat shock- and endoplasmic reticulum (ER) stress-associated genes displayed heterogeneous patterns. These differences were considered descriptive because only one pooled scRNA-seq library was generated for each workflow. These findings support gradient-free isolation as a possible alternative to Ficoll-based purification for preparing murine pancreatic islets for scRNA-seq analysis.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 5","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13600039/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149030859","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":"Ready for Safe Cancer Treatment (RESET): Protocol for a Large-Scale Randomized Controlled Trial of an Integrated Perioperative Care and Safe-Discharge Pathway in Oncological and Older Surgical Patients.","authors":"Natalia Jędruchniewicz, Hanna Klimza, Marek Zawadzki, Mariola Dwornikowska-Dąbrowska, Bernard Zając, Tomasz Klimek, Mariusz Kiszka, Mariusz Chabowski, Dorota Kamińska, Beata Jankowska-Polańska, Wojciech Witkiewicz, Małgorzata Wierzbicka","doi":"10.3390/mps9050134","DOIUrl":"10.3390/mps9050134","url":null,"abstract":"<p><p><b>Background:</b> Prolonged hospitalization, postoperative complications, and unplanned readmission remain major problems after major surgery. RESET evaluates a continuous perioperative pathway integrating individualized prehabilitation, coordinated inpatient care, enhanced-recovery principles, discharge planning, and transitional care in adults undergoing oncological surgery and patients aged 70 years or older undergoing non-oncological surgery. <b>Methods:</b> RESET is a prospective, two-center, two-arm randomized controlled study (NCT07727876). Up to 12,800 patients will be screened and approximately 8960 randomized 9:1 to RESET or standard care, including about 5740 oncological and 3220 non-oncological participants. The three primary outcomes are prolonged index hospitalization (≥14 days), unplanned readmission within 30 days after discharge, and postoperative complications within 30 days after surgery. Each outcome is evaluated separately in both clinical populations, yielding six primary hypothesis tests. Holm's procedure will control the family-wise error rate at α = 0.01 across all six tests, with endpoint- and population-specific confirmatory conclusions. Secondary outcomes include length of stay, multidomain health status, satisfaction, adherence, organizational effectiveness, and direct healthcare costs. <b>Conclusions:</b> RESET will evaluate the clinical and implementation effects of an integrated perioperative pathway across two surgical populations.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 5","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13600218/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149030968","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":"LAWS-HiC: A Locally Adaptive Weighting and Screening (LAWS) Approach to Improve Detection of Long-Range Chromatin Interactions from Hi-C Data.","authors":"Lingbo Zhou, Chang Chen, Jane Zizhen Zhao, Ming Hu, Yun Li","doi":"10.3390/mps9050135","DOIUrl":"10.3390/mps9050135","url":null,"abstract":"<p><p>Hi-C technologies are widely used to study genome-wide chromosome spatial organization. Among various Hi-C data analyses, detecting long-range chromatin interactions (i.e., 3D peak calling) is particularly critical due to its direct relevance to gene regulation. However, most existing peak callers fail to account for spatial dependencies in high-resolution (e.g., ≤10 kb) Hi-C data, often resulting in suboptimal accuracy. To address this limitation, we introduce LAWS-HiC, a novel computational method based on the Locally Adaptive Weighting and Screening (LAWS) approach. LAWS-HiC adjusts <i>p</i>-values from a standard Hi-C peak caller by incorporating local spatial dependence within topologically associating domains (TADs) in a data-driven manner. Benchmarking in two deeply sequenced Hi-C datasets from human lymphoblastoid cell line GM12878 and mouse embryonic stem cells (mESCs), LAWS-HiC consistently improves the area under the precision-recall curve (PRAUC) across varying sequencing depths, with larger gains at lower sequencing depths. At the standard Benjamini-Hochberg false discovery rate (BH-FDR) ≤ 0.05, biological feature overlap analysis confirms that LAWS-HiC calls were more strongly enriched for regulatory annotations relative to non-calls than those from alternative methods. LAWS-HiC is freely available as an R package on GitHub.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 5","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13600098/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149030914","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":"Position-Dependent Hemodynamics of Popliteal Artery Stent Grafts: The POPFLEX Study.","authors":"Christos Chronis, Konstantinos Tzirakis, Nikolaos Kontopodis, Nikolaos Galanakis, Elias Kehagias, Christos V Ioannou","doi":"10.3390/mps9050133","DOIUrl":"10.3390/mps9050133","url":null,"abstract":"<p><p><b>Background:</b> Endovascular exclusion of popliteal artery aneurysms (PAAs) with long covered stent-grafts is valuable for patients unfit for open surgery but is undermined by late stent thrombosis, even in fully scaffolded segments without kinking, a failure best clarified by computational fluid dynamics (CFD), which requires anatomically accurate 3D models-difficult to obtain in the mobile knee. <b>Methods:</b> In this prospective, single-centre study, consecutive patients undergoing endovascular PAA repair over 24 months (≥20 patients) underwent CT angiography in three knee positions (full extension, 45° and 90° flexion). Every treated limb was included, with limb as the unit of analysis. Images undergo a geometric pipeline: segmentation in 3D Slicer, then refinement in MeshLab, Meshmixer and VMTK. Meshes are converted to hexahedral volumes (ANSA Cadense) and simulated under pulsatile flow (ANSYS Fluent). Primary endpoints are time-averaged wall shear stress (TAWSS), oscillatory shear index (OSI) and relative residence time (RRT) over the stented segment, plus the surface proportion meeting thrombogenic criteria; positions are compared via linear mixed-effects models with limb nested within patient. <b>Results:</b> The protocol reproducibly yields watertight, artefact-free models preserving physiological morphology and supporting TAWSS, OSI and RRT quantification across knee positions. <b>Conclusions:</b> This protocol standardizes generation of CFD-ready popliteal geometries, enabling positional analysis of stent thrombosis.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 5","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13600194/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149030865","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":"Unveiling Distinct Developmental and Cellular Impacts of Cisplatin on Zebrafish (<i>Danio rerio</i>) Embryos: Application of a Combined Developmental Toxicity with Rapid DNA-Content Flow Cytometry Protocol.","authors":"Maximos I Leonardos, Vasileia Karavida, Anna Tsoukaneli, Dimitrios Leonardos, Yannis V Simos, Konstantinos I Tsamis, Dimitrios Peschos, Georgios S Markopoulos, Lampros Lakkas","doi":"10.3390/mps9050132","DOIUrl":"10.3390/mps9050132","url":null,"abstract":"<p><p>Rapid cellular assessment of cell-cycle perturbation can complement conventional developmental toxicology in zebrafish, but rapid DNA-content workflows developed for mammalian tissues require adaptation for whole embryos. Here, we applied an embryo-adapted version of rapid DNA-content flow cytometry analysis within a 96-h cisplatin exposure model in zebrafish (<i>Danio rerio</i>) and integrated this cellular readout with morphometric, cardiac, and neurobehavioral analysis. Dechorionated embryos were exposed to 10-400 μM cisplatin and evaluated for body length, eye surface area, heart rate, locomotor activity, thigmotaxis, touch-evoked and vibrational startle responses, and whole-embryo cell-cycle distribution. The adapted workflow combines immediate mechanical dissociation, filtration, brief propidium iodide/RNase staining, singlet gating, and direct flow cytometric acquisition. Importantly, each flow cytometry sample consisted of a single embryo, enabling DNA-content analysis at the individual-embryo level without pooling. Cisplatin produced time- and concentration-dependent growth effects and high-dose behavioral alterations, whereas heart rate was comparatively preserved. Flow cytometry detected the clearest cell-cycle redistribution at 400 μM, with a significant reduction in G0/G1 and an increase in G2/M. These findings demonstrate the practical applicability of a combined rapid embryo-adapted DNA-content workflow as a complementary cellular endpoint in a zebrafish developmental-toxicity platform and support further methodological application across experimental settings.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 5","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13600024/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149030980","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 Carnosine and Anserine in Diverse Meat Matrices via Optimized Extraction and Validated HPLC-DAD.","authors":"Chalita Kaewkhow, Chokwan Chumang, Piboon Pantu, Patraporn Luksirikul, Prapasiri Pongprayoon, Skorn Koonawootrittriron, Thanathip Suwanasopee, Tharinee Saleepoch, Sutasinee Kityakarn","doi":"10.3390/mps9050131","DOIUrl":"10.3390/mps9050131","url":null,"abstract":"<p><p>Carnosine and anserine are bioactive, histidine-containing dipeptides recognized for their antioxidant, pH-buffering, and neuroprotective functions. However, their precise quantification is frequently compromised by their high structural similarity and complex matrix-induced analytical interferences. This study developed and validated an optimized, simplified extraction protocol coupled with high-performance liquid chromatography diode array detection (HPLC-DAD) for the simultaneous determination of both dipeptides across diverse meat matrices, including Thai native black-bone chicken (Nin-Kaset), commercial broiler, pork, beef, and buffalo meat. Extraction efficiency was systematically optimized by modulating the solution pH. Under optimized conditions, the validated method demonstrated excellent linearity (1-200 µg mL^-1, r² ≥ 0.9990) with limits of detection and quantification (LOD/LOQ) values at 0.0093/0.0283 mg g^-1 for carnosine and 0.0139/0.0420 mg g^-1 for anserine. Method accuracy was confirmed by satisfactory recoveries ranging from 96.64% to 102.68%, with repeatability values (RSDs) of 0.70-14.18%. Application to real matrices revealed distinct dipeptide profiles. Pork contained the highest carnosine level (3.59 ± 0.19 mg g^-1), whereas white-feather Nin-Kaset exhibited the highest anserine level (6.77 ± 0.40 mg g^-1). Overall, the validated framework offers a rapid, cost-effective, and robust approach for routine dipeptide profiling, supporting future applications in food authentication, nutritional evaluation and functional food development.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 5","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13600070/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149031026","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 Integrated Methodological Framework for SoilHealth Assessment in Volcanic Island Agroecosystems: Indicator Selection, Analytical Workflows and Context-Sensitive Interpretation.","authors":"Sara S González-González, Mónica González-González","doi":"10.3390/mps9050129","DOIUrl":"10.3390/mps9050129","url":null,"abstract":"<p><p>Soil health assessment is increasingly used to support sustainable land management and agronomic decision-making, but generic indicator lists and universal scoring systems may be inadequate for highly reactive volcanic soils. This critical methodological review proposes an integrated methodological framework for volcanic island agroecosystems that explicitly separates analytical measurement from interpretation. The framework links assessment objectives and site context to fit-for-purpose analytical levels and classifies evidence as method-matched thresholds, local or reference benchmarks, relative or temporal comparisons, mechanistic evidence and plant-soil response validation. The reviewed evidence shows that standardized measurements do not necessarily support transferable diagnostic criteria and that volcanic-soil and andic properties can affect both analytical behavior and the functional meaning of indicator values. Rather than assigning universal scores, indicators are integrated by soil function and interpreted in relation to soil mineralogy, crop requirements, management history, and disturbance or recovery trajectories. This approach helps distinguish intrinsic volcanic-soil properties from management-induced degradation, identify functionally relevant constraints and make diagnostic confidence explicit. The framework provides a practical basis for routine monitoring, targeted diagnosis, and the development of locally validated soil health reference systems in volcanic island agriculture.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 5","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13600072/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149030916","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":"Intermittent Theta Burst Stimulation Combined with Cognitive Training in Mild Cognitive Impairment: Protocol for an 18-Week Randomised, Double-Blind, Sham-Controlled Feasibility Pilot Study (The iCog.X Boost Trial).","authors":"Rose Mery Bou Merhy, Rocco Cavaleri, Ghufran Alhassani, Vincent Oxenham, Najwa-Joelle Metri, Daniel Hochstrasser, Mark I Hohenberg, Kujan Nagaratnam, Kawaljit Singh, Genevieve Z Steiner-Lim","doi":"10.3390/mps9050130","DOIUrl":"10.3390/mps9050130","url":null,"abstract":"<p><p><b>Introduction:</b> Mild cognitive impairment (MCI) is a transitional stage between normal ageing and dementia, affecting ~35% of adults aged 70+ and carrying a 15-18% annual risk of progression. Cognitive training (CT) shows promise but outcomes are variable and transfer to daily function is unclear; intermittent theta burst stimulation (iTBS) may enhance prefrontal neuroplasticity. Few studies have evaluated whether combining CT and iTBS in MCI is practical, acceptable, and safe. <b>Methods:</b> This 18-week, randomised, double-blind, sham-controlled feasibility pilot trial will recruit 44 individuals with MCI, randomised 1:1 to CT + active iTBS or CT + sham iTBS. Participants complete 14 CT sessions over 10 weeks; accelerated iTBS is delivered in five sessions per day over five consecutive days at 75% resting motor threshold to the left DLPFC using neuronavigation, with reversed-coil sham. Primary outcomes are feasibility, acceptability, adherence, and safety; secondary (exploratory) outcomes are cognitive, functional, and psychological; tertiary (mechanistic) outcomes are EEG- and TMS-derived neurophysiological measures. The trial is not powered for efficacy: feasibility and neurophysiological outcomes will be analysed descriptively, while secondary outcomes will be analysed using linear mixed-effects models. <b>Ethics and dissemination:</b> Ethical approval has been granted by the Western Sydney University HREC (H16500); written informed consent will be obtained before enrolment. <b>Trial registration number:</b> ACTRN12625000952448.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 5","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13600087/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149030873","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":"A Single-Tube Nested PCR Method for <i>SMN1</i> Deletion Detection in Spinal Muscular Atrophy.","authors":"Ayano Kosaka, Makoto Sakima, Yoriko Noguchi, Tomoyoshi Shiroshita, Yasuhito Aoki, Yoshihiko Otsuka, Yoshihiro Bouike, Hisahide Nishio","doi":"10.3390/mps9050128","DOIUrl":"10.3390/mps9050128","url":null,"abstract":"<p><p>Spinal muscular atrophy (SMA) is a rare autosomal recessive neuromuscular disorder caused predominantly by homozygous deletion of <i>SMN1</i>, resulting in degeneration of lower motor neurons and progressive muscle weakness and atrophy. In recent years, newborn screening programs for SMA using dried blood spots and PCR-based assays have been introduced in several countries, enabling presymptomatic diagnosis and earlier initiation of therapy. However, newborn screening does not eliminate the need for diagnostic testing in routine clinical practice, because adolescents and adults with milder or ambulant phenotypes may still present only after symptom onset and may experience diagnostic delay. We therefore developed a single-tube nested PCR (STNPCR) method as a practical diagnostic approach for symptomatic patients with suspected SMA. This method enables detection of homozygous <i>SMN1</i> deletions using only standard PCR procedures and gel electrophoresis and may help identify patients with SMA who are not captured by newborn screening. Our assay using dried blood spot samples demonstrated preliminary technical feasibility in this proof-of-concept cohort, although further validation in larger independent cohorts will be required before its diagnostic utility can be established.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 5","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13600069/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149030884","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}