To avoid failure of endodontic treatment, it is important to properly seal the root canals after chemomechanical preparation in order to avoid microleakage. Typically, sealing involves the use of gutta-percha and sealing cement, the latter of which adheres to the dentin and fills the spaces between the gutta-percha and the dentin walls. Laboratory studies have been performed to detect microleakage of endodontic sealers based on various criteria, including microbiological ones; however, none guaranteed that the leakage occurred through the root canal space and not through other pathways. This article proposes a new laboratory assay to evaluate bacterial microleakage in endodontic sealers, ensuring visualization of the correct inoculum infiltration pathway. Furthermore, we present the main critical parameters and provide suggestions on how to manage them. These protocols allow the detection of bacterial microleakage in bovine teeth filled with endodontic sealer and inoculated with Enterococcus faecalis. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Bovine teeth selection and cleaning Basic Protocol 2: Bovine teeth inclusion Basic Protocol 3: Tooth standardization Basic Protocol 4: Set preparation Basic Protocol 5: Inoculum preparation Basic Protocol 6: Canal inoculation Basic Protocol 7: Set incubation.

Pseudouridine (Ψ) is a widespread RNA modification that influences RNA stability, structure, and translation. However, its role in bacterial mRNA, particularly within complex microbiomes, remains poorly defined. Here, we describe a bisulfite-based sequencing workflow coupled with a scalable computational pipeline for base-resolution, quantitative mapping of pseudouridine in microbiome transcriptomes. The protocol is optimized for low-input, high-complexity samples and includes strategies for efficient RNA extraction, ribosomal RNA depletion, bisulfite conversion, library preparation, and sequencing. The accompanying analysis pipeline enables detection and quantification of Ψ sites from chemically induced signatures, with modules for read alignment, site calling, and filtering in mixed-bacterial datasets. This approach addresses key challenges in microbiome transcriptomics, including limited biomass, high rRNA content, and community heterogeneity. The protocol can be applied to samples from diverse microbial ecosystems to generate pseudouridylation profiles, enabling investigation of pseudouridine's role in post-transcriptional regulation across microbial communities. Published 2026. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Microbiome sample collection and processing Basic Protocol 2: mRNA enrichment and bisulfite treatment Basic Protocol 3: cDNA synthesis and multiplexing of samples for sequencing Basic Protocol 4: Computational pipeline for pseudouridine analysis.

Producing recombinant bacterial proteins is essential for elucidating their structure and function, making efficient overexpression systems a key requirement. Escherichia coli is widely used for this purpose, particularly for bacterial proteins, but certain targets remain challenging to express or obtain as soluble protein. A recently developed Rhodococcus expression system has successfully produced proteins that were poorly expressed or not expressed in E. coli, highlighting the value of standardized protocols for this alternative host. Here, we present detailed methods for cloning into Rhodococcus expression vectors, transforming electrocompetent Rhodococcus cells, and performing protein expression and purification in this system. We also outline strategies to improve yields and provide troubleshooting guidance. These protocols are designed to equip protein scientists across disciplines with a robust alternative for bacterial protein production. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Cloning into a Rhodococcus expression vector Basic Protocol 2: Generation of electrocompetent Rhodococcus cells Basic Protocol 3: Transformation into a Rhodococcus host Basic Protocol 4: Recombinant protein production and purification using the Rhodococcus system.

Efficient discovery of regulatory genes and elements is essential for understanding cell identity, differentiation, and disease mechanisms. The TRIAGE methods are a set of well-established computational approaches that identify context-specific regulatory genes and prioritize regulatory elements across the genome. Previous publications have described the development of these algorithms, their benchmarking, and biological applications. Here, we provide step-by-step protocols for applying the TRIAGE methods to identify regulatory drivers from diverse input types, including gene expression matrices, gene lists, and genomic loci. It covers analyses of both bulk and single-cell RNA-seq datasets and enables genome-wide interrogation of regulatory elements at single-base resolution. The analysis is efficient, typically requiring <30 min of computation time on a personal computer. In addition to the step-by-step description of the TRIAGE analysis workflow, we provide the TRIAGE toolkit, available as both an R package and a Python implementation, to support flexible and scalable regulatory analysis across platforms. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Prioritization of regulatory genes from bulk RNA-seq data Basic Protocol 2: Identification of cell populations and regulatory genes in single-cell RNA-seq data Basic Protocol 3: Prioritization of regulatory long noncoding RNAs Basic Protocol 4: Prioritization of functional genetic variants from eQTL data Alternate Protocol: Python-based implementation of the TRIAGE workflow for regulatory gene and element prioritization Support Protocol: Preparing a normalized expression matrix from bulk RNA-seq count data.

Adolescents face significant barriers in accessing reproductive health services due to fears of privacy breaches and social stigma. In primary healthcare facilities, healthcare professionals are often trapped in a dilemma between the obligation to maintain medical confidentiality based on professional ethics and national regulatory demands requiring parental involvement for underage patients. This study aims to analyze the practice of protecting adolescent patient data privacy at Mardi Saras Primary Clinic and identify the gap between practical implementation and the norms of medical ethics and applicable legal regulations. This study employs a socio-legal research method with a qualitative approach. Data were collected through in-depth interviews with doctors and nurses, observation of service procedures, and study of the clinic's Standard Operating Procedures. The results indicate that although Mardi Saras Primary Clinic has implemented basic confidentiality protocols such as closed consultation rooms, there are substantial weaknesses in informed consent management, which still heavily relies on parental presence for all adolescent cases. This practice is driven by healthcare professionals' fear of legal risks, thereby ignoring the principle of developing autonomy in adolescents. These findings indicate a disparity between rigid administrative compliance and the spirit of protecting adolescent health rights within national regulations. The implication of this study underscores the urgency of developing specific SOPs that are sensitive to adolescent rights, as well as the need for continuous training for healthcare professionals regarding the legal and ethical boundaries of maintaining confidentiality for underage patients without compromising legal safety aspects.

Neurospora crassa is a well-established model organism for studying biological timing and light-regulated development. The Protoperithecia Assay (PPA) is a plate-based imaging method for quantifying photoperiodic responses by measuring protoperithecia formation as a developmental output. The goal of this protocol is to provide a reproducible and scalable workflow for assessing how defined light-dark cycles influence fungal sexual development. In this assay, fungal cultures are grown on synthetic crossing medium under controlled photoperiod conditions, followed by standardized removal of surface conidia to improve visualization. Protoperithecia are quantified by manual image-based counting, and average counts per plate are used for statistical comparisons across photoperiod conditions. Unlike prior approaches, the PPA integrates standardized culture conditions, controlled photoperiod exposure, and quadrant-based imaging to enable reproducible quantitative comparisons across strains. This workflow extends beyond a basic protocol by establishing a consistent and scalable framework for measuring photoperiod-dependent developmental output under defined experimental conditions. The method requires approximately one week from inoculation to analysis and can be applied across multiple strains under defined environmental conditions. The PPA provides a simple and accessible platform for comparative analysis of photoperiod-dependent development in fungi under controlled laboratory conditions.

Nicotinamide adenine dinucleotide (NAD+) is an essential metabolite involved in numerous biological processes, functioning both as a redox cofactor in cellular energy metabolism and as a co-substrate for NAD+-utilizing enzymes, including sirtuins and poly(ADP-ribose) polymerases. Given its central role in maintaining physiological homeostasis and its involvement in a wide range of disease processes, there is growing interest in developing NAD+ and its analogs as chemical probes and potential therapeutic agents. However, the chemical synthesis of NAD+ analogs is often hampered by low yields, poor stereoselectivity, and the formation of undesired byproducts. In contrast, enzymatic synthesis offers a more efficient and selective strategy using readily available starting materials under mild conditions. Herein, we report a two-step enzymatic strategy for the synthesis of NAD+. In the first step, a ten-enzyme coupled reaction cascade converts glucose into nicotinic acid adenine dinucleotide (NaAD). In the second step, the isolated NaAD is subsequently converted to NAD+ by NAD+ synthetase. This modular enzymatic platform provides an efficient, scalable approach to NAD+ production and establishes a versatile foundation for the biosynthesis of NAD+ analogs for biochemical and biomedical applications. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Transformation of plasmid DNA into Rosetta(DE3) cells Basic Protocol 2: Recombinant expression, purification, and activity assessment of NMA1 Basic Protocol 3: Recombinant expression, purification, and activity assessment of Ta1145 Basic Protocol 4: Recombinant expression, purification, and activity assessment of PrsA Basic Protocol 5: Two-step enzymatic synthesis of NAD.

The limited penetration of chemotherapeutic agents into solid tumors remains a major obstacle to effective cancer treatment and is strongly influenced by the extracellular matrix (ECM). Three-dimensional (3D) tumor spheroids derived from primary tumor cells (PTCs) represent a valuable in vitro model to study how ECM composition and organization regulate drug distribution and cytotoxicity with accurate physiological relevance. By recapitulating key features of the tumor microenvironment, including endogenous collagen deposition and diffusion-limited drug accessibility, these models enable mechanistic investigation of microenvironment-driven drug resistance. Here, we describe a set of integrated protocols to evaluate the impact of ECM remodeling on chemotherapeutic response in primary breast tumor spheroids. Spheroids are generated from isolated primary tumor cells and subjected to controlled enzymatic degradation of collagen-rich ECM using ultrapure recombinant collagenases. As a model chemotherapeutic agent, doxorubicin is employed thanks to its widespread clinical use, well-characterized cytotoxic mechanism, and intrinsic fluorescence, which allows direct visualization of drug uptake and spatial distribution by confocal microscopy. In parallel, drug-induced cytotoxicity is quantified using a luminescent 3D viability assay. Together, these protocols provide a reproducible and accessible platform for investigating ECM-mediated barriers to drug delivery and toxicity in 3D tumor models. This framework facilitates the implementation of physiologically relevant assays to study drug efficacy and therapeutic resistance in solid tumors. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Induction of breast cancer, isolation of tumor primary cells, and generation 3D spheroids Support Protocol: Culture and storage of PTCs Basic Protocol 2: Evaluation of collagen expression and enzymatic degradation in primary tumor spheroids by confocal microscopy Basic Protocol 3: Enzymatic degradation of the endogenous matrix collagen in primary tumor spheroids using recombinant collagenases Basic Protocol 4: Assessment of doxorubicin uptake in spheroids by confocal microscopy Basic Protocol 5: Assessment of cell viability in doxorubicin-treated spheroids using CellTiter-Glo 3D assay.

Mitochondrial calcium (mCa2+) homeostasis promotes oxidative metabolism within the physiological range; however, dysregulation can trigger necrotic cell death in diseases such as cardiac ischemia-reperfusion injury, muscular dystrophy, and neurodegenerative disorders, including Alzheimer's disease. It is widely understood that mitochondria exhibit rapid Ca2+ uptake primarily mediated by the mitochondrial calcium uniporter (MCU) complex, and that Ca2+ is exported via a combination of Na+/Ca2+ and H+/Ca2+ exchange processes. However, the proteins which mediate mCa2+ transport have only been partially identified. A particular challenge in determining which proteins mediate mCa2+ efflux and their relative contributions to mCa2+ homeostasis is the lack of a clear, reproducible assay for mCa2+ efflux applicable across genotypes. Here, we provide instructions for an optimized fluorometric method to measure mCa2+ efflux in isolated mitochondria that establishes robust Ca2+ efflux signals, differentiates between total and Na+-independent Ca2+ efflux modalities, and generates highly reproducible data, allowing comparisons across tissues and genotypes. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Total Calcium Efflux Assay in Isolated Mitochondria Alternate Protocol 1: Na+-Independent Calcium Efflux Assay in Isolated Mitochondria Support Protocol 1: Isolation of Cardiac Mitochondria Support Protocol 2: Isolation of Skeletal Muscle Mitochondria Support Protocol 3: Isolation of Liver Mitochondria Support Protocol 4: Isolation of Cell Line Mitochondria Basic Protocol 2: Analysis of Calcium Efflux.

Jupyter Notebooks have transformed the way we conduct computational research thanks to a versatile platform that combines code, annotations, plots, and a lightweight format that enables versioning. However, steps for installing dependencies and downloading data for a specific notebook may require significant time and failure to establish identical environments can compromise reproducibility of environments. Deploying a JupyterHub server not only provides a containerized environment for each notebook to ensure fast execution and exact reproducibility but also provides a computational engine that makes these applications available from any device. By providing all the steps for deploying a JupyterHub server, and providing our experience deploying and maintaining Netbooks, a server for academic investigations in network biology, we want to encourage other groups to deploy JupyterHub servers for their own computational work and to recognize their value in improving reproducibility, accelerating manuscript review, and supporting education. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Setting up a JupyterHub server Support Protocol 1: Compiling a collection of Jupyter Notebooks Support Protocol 2: Securing HTTPS access Support Protocol 3: Adding parameters for JupyterHub Support Protocol 4: Data management for JupyterHub Support Protocol 5: Customizing the website homepage Support Protocol 6: Importing/Exporting conda environments Alternate Protocol: Running a JupyterHub server as a virtual machine.

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is low-cost, widely available, and mainly used to assess cell viability as a function of cellular oxidoreductase enzyme-dependent conversion of the water-soluble tetrazolium salt to insoluble purple formazan. Although MTT is often associated with mitochondrial redox activity, extra-mitochondrial MTT reduction has been identified in several cell types, shaping the assay towards a total oxidoreductase/metabolic activity measurement. Standard MTT protocols were developed for adherent monolayer cells and perform poorly with suspension cells, as medium removal, cell and formazan loss, aggregate formation, and incomplete formazan solubilization result in data with high variability and nonlinearity. To overcome these limitations, here we describe an easy, inexpensive, and optimized MTT protocol for evaluating the metabolic activity in human spermatozoa. Key improvements in cell number optimization, compound and medium non-interference validation, post-assay cell preservation, and homogenous formazan solubilization resulted in a reproducible and linear protocol compatible with conventional microplate readers. This method quantifies spermatozoa metabolic activity with improved precision across technical replicates and can be directly applicable to toxicology screening, spermatozoa quality assessment, and fertility research focused on spermatozoa bioenergetics. Furthermore, although optimized for human spermatozoa, the workflow described in this protocol is readily adaptable to other suspension cell types following simple cell-specific calibration. © 2026 Wiley Periodicals LLC. Basic Protocol: Quantitative evaluation of human spermatozoa metabolic activity using MTT.

Concatenating Original Duplex for Error Correction (CODEC) is a duplex sequencing library preparation method that physically links the Watson and Crick strands of individual DNA molecules. Despite growing interest in cost-effective duplex sequencing such as CODEC, broader adoption can be limited by the lack of detailed, step-by-step wet-lab workflows. Here, we describe an experimentally validated protocol for preparing CODEC libraries from genomic DNA that are compatible with standard Illumina platforms. The protocol incorporates blunt-end fragmentation, dideoxy base-blocked A-tailing, CODEC adapter ligation, strand-displacing extension, and library amplification, with quality-control checkpoints, critical parameters, and troubleshooting guidance. In addition, we provide an optional matched germline reference library preparation protocol to support somatic variant calling. This workflow is intended to facilitate reproducible implementation of CODEC in molecular biology laboratories for research applications requiring accurate detection of low-frequency somatic variants. © 2026 Wiley Periodicals LLC. Support Protocol 1: CODEC oligonucleotide ordering and synthesis Support Protocol 2: CODEC quadruplex adapter synthesis Basic Protocol 1: CODEC library preparation from genomic DNA Support Protocol 3: AMPure XP/SPRIselect bead cleanup Basic Protocol 2: Matched germline reference library preparation from genomic DNA.

Self-amplifying RNA (saRNA) derived from alphavirus replicons enables robust intracellular RNA amplification and high-level protein expression at substantially lower doses than nonreplicating messenger RNA (mRNA) platforms. Venezuelan equine encephalitis virus (VEEV)-based replicons lacking the viral structural genes are among the most extensively characterized saRNA backbones, combining efficient cytoplasmic replication with a favorable safety profile. However, incorporating chemical nucleoside modifications such as N1-methylpseudouridine (m1ψ), now standard in conventional mRNA workflows, can directly impact replicase activity and hinder RNA amplification, necessitating backbone-specific optimization. Recent studies have shown that incorporation of 5-methylcytidine (m5C) preserves VEEV saRNA replication while reducing innate immune activation and improving expression durability. In an independent study, phosphatase treatment to remove residual 5'-triphosphates was shown to enhance saRNA functionality. Building on these findings, here we describe a streamlined protocol for generating m5C-modified VEEV-based saRNA using a single-step in vitro transcription (IVT) strategy. This protocol utilizes PCR-generated DNA templates with an encoded poly(A) tail, CleanCap AU for co-transcriptional capping, and post-transcriptional phosphatase treatment to minimize immunostimulatory RNA species. Finally, the RNA was purified using the phenol-chloroform-isoamyl alcohol method and functionally evaluated by transfection into HEK293T cells. This protocol provides a reproducible framework for producing capped, tailed, and chemically modified saRNA suitable for downstream functional and translational studies. © 2026 Wiley Periodicals LLC. Basic Protocol 1: IVT and purification of saRNA with modified nucleotides Basic Protocol 2: Functional assessment of IVT-generated saRNA in HEK293T Cells.

Genome editing can enhance basic research and enable industrial applications of green algae. Here, we present an affordable, broadly applicable workflow for genome editing in the unicellular green alga Euglena gracilis using Cas9 nucleases. This method retains high editing efficiency while significantly lowering technical barriers. Unlike previous approaches that required specialized equipment, this protocol can be performed using a general-purpose laboratory electroporator and a simplified clonal isolation procedure without the need for specialized micromanipulation devices. This protocol is compatible with a range of editing outcomes, such as targeted deletions and precise base substitutions, enabling more widespread genome editing in Euglena. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culture of Euglena gracilis Basic Protocol 2: sgRNA synthesis Basic protocol 3: Transformation Basic protocol 4: Genotyping.

Alveolar epithelial cell type 1 (AT1) and type 2 (AT2) cells make up the saccular gas exchange units of the lung, called alveoli. Formation of alveoli during lung development accounts for the expansive surface area of the lung, allowing for proper respiration and delivery of oxygen to the body. Due to their delicate structure, alveoli are susceptible to injury caused by environmental exposures, such as inhaled cigarette smoke and heavy metals. Chronic exposure to these toxicants can exacerbate preexisting conditions such as asthma or contribute to the progression of lung diseases, such as chronic obstructive pulmonary disease (COPD), interstitial lung disease (ILD), and cancer. AT2 cells play a key role in injury repair and regeneration of the distal lung and there is widespread interest in their use as cellular models for lung disease in vitro. However, while immortalized cell lines derived from airway epithelial cells were successfully generated decades ago, human adult alveolar epithelial cell lines have proven to be more difficult to establish due to their limited proliferative capacity. Here we describe an extensive end-to-end method for deriving immortalized alveolar epithelial cells (AECs), termed "AEC-tLgT cells," from normal human lung tissue. We first outline a detailed procedure to isolate AT2 cells. We then outline our optimized method for immortalizing AT2 cells to generate polyclonal cell lines. We next describe a three-dimensional co-culture system to induce lung organoid formation from immortalized AT2 cell lines. Finally, we describe a procedure for studying cigarette smoke and nickel exposure using immortalized AT2 cell lines to investigate environmental toxicity. These protocols will enable users to generate AEC models from donors with defined genetic, demographic, and clinical backgrounds, facilitating the study of differential susceptibility to environmental exposures and risk for distal lung diseases. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation and purification of human AT2 cells Basic Protocol 2: Immortalization of AT2 cells and maintenance of resulting AEC lines Basic Protocol 3: Determination of organoid formation ability Basic Protocol 4: Treatment of immortalized AEC lines to study environmental exposures.

Protein language models (PLMs) trained solely on sequence data have significantly advanced our understanding of protein biology and achieved remarkable performance in protein prediction tasks. However, their lack of three-dimensional (3D) structural features limits their predictive power in applications that rely heavily on 3D conformation. To address this limitation, we developed two structure-aware PLMs, S-PLM1 and S-PLM2, that employ multi-view contrastive learning to align protein sequences with their 3D structures in a unified latent space. S-PLM1 represents structural information using contact maps encoded by a pretrained Swin-Transformer, while S-PLM2 directly encodes 3D backbone coordinates through a Geometric Vector Perceptron (GVP)-based model. The paired sequence-structure data were obtained from AlphaFoldDB. For both models, we designed efficient tuning strategies that enable optimal performance with minimal computational cost. Here, we present detailed protocols for adapting S-PLM1 and S-PLM2 for diverse protein applications. The protocols provide step-by-step guidance on generating structure-aware representations from S-PLMs, fine-tuning them for various protein prediction tasks, and using S-PLM2 to produce structure embeddings for structure-based downstream analyses. We also provide source code and Google Colab implementations for easy customization and deployment. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Generating structure-aware representations of protein sequences Basic Protocol 2: Efficient tuning of structure-aware protein language models for diverse protein applications Basic Protocol 3: Using S-PLM2 to generate protein structure representations and conduct structure-based clustering Support Protocol: Google Colab quick start notebooks.

Proteolysis-targeting chimeras (PROTACs) have emerged as a promising strategy for the selective degradation of intracellular proteins by harnessing the ubiquitin-proteasome system. However, the development of PROTACs is often limited by the availability of high-affinity ligands, particularly for targets that are considered difficult to drug, such as transcription factors. In this context, aptamers, which are synthetic nucleic acid molecules capable of high affinity and specificity toward their targets, have attracted increasing attention as alternative binding moieties for PROTAC design. This protocol reports the synthesis of (1) a chimeric molecule in which a nucleic acid aptamer capable of binding to estrogen receptor alpha (ERα) and an E3 ubiquitin ligase ligand is linked via a linker, and (2) a phosphorothioate (PS)-modified analogue to enhance nuclease resistance and cellular uptake. A series of aptamer-PROTACs with various PS modification patterns were synthesized and conjugated to cereblon ligands via copper-catalyzed click chemistry. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Solid-phase synthesis of DNA aptamer Support Protocol 1: Preparation of 5'-hexynyl phosphoramidite Support Protocol 2: Preparation of CuI·P(OEt)3 Basic Protocol 2: Synthesis of DNA aptamer-based PROTAC Alternate Protocol: Synthesis of phosphorothioate-modified DNA-based PROTAC.

Quantitative RNA imaging in large plant tissues has historically been challenging because of limited spatial resolution, low signal-to-noise ratios, and the largely qualitative nature of traditional RNA in situ hybridization methods. Hybridization chain reaction-RNA fluorescence in situ hybridization (HCR RNA-FISH) coupled with high-resolution microscopy enables sensitive detection of RNA molecules at cellular resolution. However, quantitative approaches that combine improved tissue accessibility with robust computational pipelines for single-cell transcript quantification in plants remain limited. Here, we present a quantitative HCR RNA-FISH protocol for the developing maize inflorescence. We describe a 4-day workflow that includes fixation, agarose immobilization, vibratome sectioning, probe hybridization, amplification, and mounting and enables multiplexed detection of transcripts at the cellular level in maize ear and tassel primordia. In addition, we provide a Python-based image analysis pipeline for (i) cell segmentation, (ii) RNA spot quantification, (iii) assignment of spots to cells, and (iv) data representation. The scripts can be easily run on Jupyter notebooks and are available on GitHub. Overall, this protocol highlights the importance of integrating robust imaging strategies with quantitative and reproducible data analysis frameworks to extract biologically meaningful insights from imaging data. © 2026 Wiley Periodicals LLC. Basic Protocol: Quantitative RNA imaging in sections of maize ear and tassel primordia using HCR RNA-FISH.

This article describes a validated and optimized sequence-specific primer polymerase chain reaction (PCR-SSP) protocol for genotyping the human TLR9 promoter polymorphism rs187084 (-1486T>C). Promoter single nucleotide polymorphisms (SNPs) may influence transcriptional regulation and immune signaling. The rs187084 variant has been associated with altered TLR9 expression and susceptibility to infectious, inflammatory, and neoplastic diseases. Although Sanger sequencing provides high analytical accuracy, it is labor-intensive and costly for large cohorts. TaqMan allelic discrimination assays require fluorescent probes and real-time PCR instrumentation, increasing cost per sample. High-resolution melting (HRM) analysis depends on specialized melting platforms and curve interpretation expertise. The PCR-SSP strategy described here provides a robust and accessible approach based on conventional thermocyclers and standard agarose gel electrophoresis, without the need for specialized instrumentation or probe-based chemistries. Allele discrimination is achieved using reverse primers differing at the 3' terminal nucleotide. A 430-bp human growth hormone (HGH) fragment is co-amplified as internal control. Optimization of MgCl2 concentration, primer ratios, and annealing temperature ensured specificity, sensitivity, and reproducibility. This protocol is suitable for genetic association studies and population-based analyses of TLR9 rs187084. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: PCR-SSP genotyping of TLR9 rs187084 Support Protocol: Genomic DNA extraction from whole blood by the salting-out method.

Single nucleotide polymorphisms (SNPs) can modify protein expression or function, influencing susceptibility to various diseases. Interleukin-22 (IL-22), involved in epithelial barrier homeostasis and tissue repair, has shown complex roles in autoimmune, infectious, and inflammatory conditions. This article describes a standardized protocol for genotyping the IL-22 SNPs rs2227473 (C>T) and rs2227513 (A>G) using sequence-specific primer polymerase chain reaction (PCR-SSP), with validation by Sanger sequencing. The protocol includes optimization of annealing temperature, MgCl2 concentration, primer balance, deoxynucleotide triphosphates (dNTPs), and DNA input, using the human growth hormone (HGH) gene as an internal control. Amplified products are analyzed by agarose gel electrophoresis. Complete concordance with Sanger sequencing confirms the reliability of the method. This protocol provides a rapid, cost-effective, and reproducible approach for SNP genotyping, suitable for large-scale studies. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: PCR-SSP genotyping of IL-22 SNPs rs2227473 and rs2227513 Support Protocol: Validation of genotypes by Sanger sequencing.