分子生物学

Zebrafish are a powerful model for investigating vascular and lymphatic biology due to their genetic tractability and optical transparency. While translating ribosome affinity purification (TRAP) has been widely applied in other systems, its application in zebrafish has remained limited. Here, we present an optimized TRAP protocol for isolating ribosome-associated mRNAs from endothelial cells in vivo, without the need for cell dissociation or sorting. Using a novel transgenic zebrafish line, which expresses HA-tagged Rpl10a under the mrc1a promoter, we enriched actively translating endothelial transcripts. Differential expression analysis revealed robust upregulation of vascular and lymphatic genes including flt4, kdrl, and lyve1b. This approach captures the endothelial cell translatome with high specificity and offers a robust platform for investigating the molecular mechanisms of endothelial biology under genetic, environmental, or toxicological perturbations.

Genome-walking protocols have been extensively used to clone unknown genomic sequences next to known DNAs. Existing genome-walking protocols need further improvement in methodological specificity or operation. Here, we describe a novel genome-walking protocol based on fusion primer–driven racket PCR (FPR-PCR). FPR-PCR involves four sequence-specific oligos (SSO), SSO1, SSO2, SSO3, and SSO4, which are sequentially chosen from known DNA in the direction 5’→3’. The fusion primer, mediating primary FPR-PCR, is generated by attaching SSO3 to the 5’ end of SSO1. The SSO3 encourages the target DNA of primary PCR to form a racket-like structure by mediating intra-strand annealing. SSO2 and SSO4 are directly used as sequence-specific primers (SSP) in secondary FPR-PCR, which selectively amplifies this racket-like DNA. This protocol was verified by cloning several unknown genomic sequences. Compared to traditional PCRs, FPR-PCR offers the advantages of higher specificity and fewer rounds, primarily attributed to the omission of arbitrary walking primers typically required in traditional methods.

Genome walking is a classical molecular biology technique used to amplify unknown regions flanking known DNA sequences. Genome walking holds a vital position in the areas associated with molecular biology. However, existing genome-walking protocols still face issues in experimental operation or methodological specificity. Here, we propose a novel genome-walking protocol based on bridging PCR. The critical factor of this protocol is the use of a bridging primer, which is made by attaching an oligomer (or tail primer sequence) to the 5′ end of the walker primer 5′ region. When the bridging primer anneals to the walker primer site, this site will elongate along the tail of the bridging primer. The non-target product (the main contributor to background in genome walking), defined by the walker primer, is lengthened at both ends. In the next PCR(s), the annealing between the two lengthened ends is easier than the annealing between them and the shorter tail primer. As a result, this non-target product is eliminated without affecting target amplification.

Preserving biological samples in the field is essential for ensuring high-quality nucleic acid extraction and reliable downstream molecular analyses. Broadly, two main preservation strategies are available: physical preservation, such as flash freezing in liquid nitrogen, which halts enzymatic activity by rapid cooling, and chemical preservation, using stabilizing reagents that inactivate nucleases and protect nucleic acids even at ambient temperatures. This protocol presents a comparative approach using liquid nitrogen and a commercial stabilizing reagent (DNA/RNA Shield, Zymo Research) to preserve tissue from five marine invertebrate species: two cold-water corals, two sponges, and one bivalve. Samples preserved by each method were processed with the AllPrep DNA/RNA Mini kit (Qiagen) to extract both RNA and DNA. RNA quality was assessed using RNA Integrity Number (RIN) scores. The stabilizing reagent preserved high-quality RNA in sponge and bivalve samples but did not prevent RNA degradation in coral tissues, which showed lower RIN scores compared to those preserved in liquid nitrogen. DNA yields were also consistently lower in tissues preserved with DNA/RNA Shield across all species. These findings suggest that DNA/RNA Shield can be a viable alternative to liquid nitrogen for some marine invertebrates, particularly in field conditions where cryopreservation is impractical. However, for cold-water corals, liquid nitrogen remains essential to ensure RNA integrity for transcriptomic analyses and other sensitive molecular applications (e.g., RT-qPCR).

Protein phosphorylation is a dynamic post-translational modification that regulates fundamental processes, including signal transduction, cell proliferation, differentiation, and effector function of immune cells. The Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway is a key mediator of cytokine responses, essential for maintaining immune cell homeostasis and determining cell fate across diverse immune subsets. Dysregulation of JAK/STAT signaling has been linked to a broad spectrum of pathologies, including monogenic immune disorders, autoimmunity, and cancer. Platforms facilitating single-cell analysis of protein phosphorylation offer the ability to reveal subtle signaling defects and dissect the pleiotropy in cellular composition and phosphorylation status, providing insights into immune phenotype and function, while identifying potential therapeutic targets. While an application of cytometry-by-time-of-flight, termed phospho-CyTOF, has proven invaluable for studying protein phosphorylation in cryopreserved peripheral blood mononuclear cells (cPBMCs), its application is limited by cell loss and signaling artifacts stemming from isolation and cryopreservation. Conversely, whole blood (WB) approaches, preserving the native immune cell composition and signaling context, offer a more physiological representation but necessitate robust and consistent protocols for broad application. Herein, we present optimized dual phospho-CyTOF workflows tailored for both cPBMCs and whole blood, building upon established protocols for cytokine stimulation of both samples. These workflows facilitate comprehensive, high-dimensional profiling of JAK/STAT signaling in response to pleiotropic cytokines such as Type I interferons (IFN-α), Type II interferons (IFN-γ), and Interleukin-21 (IL-21). By leveraging CyTOF's capacity for high-dimensional profiling using pure heavy metal–labeled antibodies, these protocols aim to identify pathway-specific alterations in STAT phosphorylation across major immune subsets that may be overlooked by traditional flow cytometry. Together, these optimized dual workflows provide scalable, translationally relevant tools for dissecting the subtle and differential JAK/STAT-driven immune responses in both clinical and research settings, while also being compatible with the simultaneous assessment of crosstalk with alternative immune cell signaling pathways.

Real-time quantitative PCR (qPCR) is a pivotal technique for analyzing gene expression and DNA copy number variations. However, the limited availability of user-friendly software tools for qPCR data analysis presents a significant challenge for experimental biologists with limited computational skills. To address this issue, we developed Click-qPCR, a user-friendly and web-based Shiny application for qPCR data analysis. Click-qPCR streamlines ΔCq and ΔΔCq calculations using user-uploaded CSV data files. The interactive interface of the application allows users to select genes and experimental groups and perform Welch’s t tests and one-way analysis of variance with Dunnett’s post-hoc test for pairwise and multi-group comparisons, respectively. Results are visualized via interactive bar plots (mean ± standard deviation with individual data points) and can be downloaded as publication-quality images, along with summary statistics. Click-qPCR empowers researchers to efficiently process, interpret, and visualize qPCR data regardless of their programming experience, thereby facilitating routine analysis tasks. Click-qPCR Shiny application is available at https://kubo-azu.shinyapps.io/Click-qPCR/, while its source code and user guide are available at https://github.com/kubo-azu/Click-qPCR.

Telomere length maintenance is strongly linked to cellular aging, as telomeres progressively shorten with each cell division. This phenomenon is well-documented in mitotic, or dividing, cells. However, neurons are post-mitotic and do not undergo mitosis, meaning they lack the classical mechanisms through which telomere shortening occurs. Despite this, neurons retain telomeres that protect chromosomal ends. The role of telomeres in neurons has gained interest, particularly in the context of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), where aging is a major risk factor. This has sparked interest in investigating telomere maintenance mechanisms in post-mitotic neurons. Nevertheless, most existing telomere analysis techniques were developed for and optimized using mitotic cells, posing challenges for studying telomeres in non-dividing neuronal cells. Thus, this protocol adapts an already established technique, the combined immunofluorescence and telomere fluorescent in situ hybridization (IF-FISH) on mitotic cells to study the processes occurring at telomeres in cortical neurons of the mouse ALS transgenic model, TDP-43 rNLS. Specifically, it determines the occurrence of DNA damage and the alternative lengthening of telomeres (ALT) mechanism through simultaneous labeling of the DNA damage marker, γH2AX, or the ALT marker, promyelocytic leukemia (PML) protein, together with telomeres. Therefore, the protocol enables the visualization of DNA damage (γH2AX) or the ALT marker (PML) concurrently with telomeres. This technique can be successfully applied to brain tissue and enables the investigation of telomeres specifically in cortical neurons, rather than in bulk tissue, offering a significant advantage over Southern blot or qPCR-based techniques.

DNA methylation is a fundamental epigenetic mark with critical roles in epigenetic regulation, development, and genome stability across diverse organisms. Whole genome bisulfite sequencing (WGBS) enables single-base resolution mapping of cytosine methylation patterns and has become a standard method in epigenomics. This protocol provides a detailed, step-by-step workflow for WGBS library construction starting from genomic DNA. It includes steps of RNaseA treatment, DNA shearing, end-repair and A-tailing, adapter ligation, bisulfite conversion, library amplification, and quantification. Notably, the method uses self-prepared reagents and customizable index systems, avoiding the constraints of commercial library preparation kits. This flexibility supports cost-effective, scalable methylome profiling, suitable for diverse experimental designs, including high-throughput multiplexed sequencing.

The RNA-guided Cas enzyme specifically cuts chromosomes and introduces a targeted double-strand break, facilitating multiple kinds of genome editing, including gene deletion, insertion, and replacement. Caulobacter crescentus and its relatives, such as Agrobacterium fabrum and Sinorhizobium meliloti, have been widely studied for industrial, agricultural, and biomedical applications; however, their genetic manipulations are usually characterized as time-consuming and labor-intensive. C. crescentus and its relatives are known to be CRISPR/Cas-recalcitrant organisms due to intrinsic limitations of SpCas9 expression and possible CRISPR escapes. By fusing a reporting gene to the C terminus of SpCas9M and precisely manipulating the expression of SpCas9M, we developed a CRISPR/SpCas9M-reporting system and achieved efficient genome editing in C. crescentus and relatives. Here, we describe a protocol for rapid, marker-less, and convenient gene deletion by using the CRISPR/SpCas9M-reporting system in C. crescentus, as an example.

N⁶-methyladenosine (m⁶A) is the most abundant internal modification in mRNA and is regulated primarily by the balance between the METTL3 methylase complex and two demethylases, FTO (fat mass and obesity-associated protein) and ALKBH5 (α-ketoglutarate-dependent dioxygenase alkB homolog). Reflecting this prevalence, m⁶A participates in virtually every step of RNA metabolism, influencing a wide range of physiological and pathological processes. The first step in studying m⁶A is genome-wide mapping, typically performed by m⁶A-seq, which sequences RNA fragments immunoprecipitated with an m⁶A-specific antibody. This is followed by identification of RRACH motifs (R = A or G; H = A, C, or U) within these sequences, with m⁶A being located at the third nucleotide. The second step involves mutating the putative m⁶A sites to establish a causal link between the modification and downstream biological effects. Since the mapping step has been covered in several detailed protocols, this article focuses on the second step—mutagenesis of RRACH motifs and subsequent functional analysis of the mutations by ectopic expression. The 3′ untranslated region (UTR) of the mouse Runx2 gene is used as an example. The mutant and wild-type sequences are inserted into a luciferase reporter vector and transfected into 293FT cells to evaluate how loss of m⁶A affects luciferase protein levels. The same reporter plasmids are also used in an RNA stability assay with a transcription inhibitor. Although site-specific demethylation of endogenous mRNA would be preferable, it remains technically challenging despite many attempts. Thus, ectopic expression of the mutated target gene remains a widely used and practical alternative.