分子生物学

Immunofluorescence staining is a technique that permits the visualization of components of various cell preparations. Manchette, a transient structure that is only present in elongating spermatids, is involved in intra-manchette transport (IMT) for sperm flagella formation. Sperm flagella are assembled by intra-flagellar transport (IFT). Due to the big complexes formed by IMT and IFT components, it has been challenging to visualize these components in tissue sections. This is because the proteins that make up these complexes overlap with each other. Testicular tissue is digested by a combination of DNase I and Collagenase IV enzymes and fixed by paraformaldehyde and sucrose. After permeabilization with Triton X-100, testicular cells are incubated with specific antibodies to detect the components in the manchette and developing sperm tails. This method allows for cell type–specific resolution without interference from surrounding cells like Sertoli, Leydig, or peritubular myoid cells. Additionally, isolated cells produce cleaner immunofluorescence signals compared to other methods like tissue section/whole mount, making this method the best fit for visualizing protein localization in germ cells when spatial context is not being considered. Hence, this protocol provides the detailed methodology for isolating male mice germ cells for antibody-targeted immunofluorescence assay for confocal/fluorescence microscopy.

Osteoarthritis (OA) is the primary cause of joint impairment, particularly in the knee. The prevalence of OA has significantly increased, with knee OA being a major contributor whose pathogenesis remains unknown. Articular cartilage and the synovium play critical roles in OA, but extracting high-quality RNA from these tissues is challenging because of the high extracellular matrix content and low cellularity. This study aimed to identify the most suitable RNA isolation method for obtaining high-quality RNA from microquantities of guinea pig cartilage and synovial tissues, a relevant model for idiopathic OA. We compared the traditional TRIzol^® method with modifications to spin column–based methods (TRIspin-TRIzol^®/RNeasy^TM, RNeasy^TM kit, RNAqueous^TM kit, and Quick-RNA^TM Miniprep Plus kit), and an optimized RNA isolation protocol was developed to increase RNA yield and purity. The procedure involved meticulous sample collection, specialized tissue processing, and measures to minimize RNA degradation. RNA quality was assessed via spectrophotometry and RT–qPCR. The results demonstrated that among the tested methods, the Quick-RNA^TM Miniprep Plus kit with proteinase K treatment yielded the highest RNA purity, with A_260:280 ratios ranging from 1.9 to 2.0 and A_260:230 ratios between 1.6 and 2.0, indicating minimal to no salt contamination and RNA concentrations up to 240 ng/μL from ⁓20 mg of tissue. The preparation, storage, homogenization process, and choice of RNA isolation method are all critical factors in obtaining high-purity RNA from guinea pig cartilage and synovial tissues. Our developed protocol significantly enhances RNA quality and purity from micro-quantities of tissue, making it particularly effective for RTqPCR in resource-limited settings. Further refinements can potentially increase RNA yield and purity, but this protocol facilitates accurate gene expression analyses, contributing to a better understanding of OA pathogenesis and the development of therapeutic strategies.

Ubiquitination is a post-translational protein modification that regulates a vast majority of processes during protein homeostasis. The covalent attachment of ubiquitin is a highly regulated process carried out by the sequential action of the three enzymes E1, E2, and E3. E3 ligases share a dual function of 1) transferring covalently attached ubiquitin from the catalytic cysteine of E2 (E2~Ub) to the substrate and 2) providing substrate specificity. Our current knowledge of their individual substrate pools is incomplete due to the difficult capture of these transient substrate–E3 ligase interactions. Here, we present an efficient protocol that enables the selective biotinylation of substrates of a given ubiquitin ligase. In brief, the candidate E3 ligase is fused to the biotin ligase BirA and ubiquitin to a biotin acceptor peptide, an Avi-tag variant (-2) AP. Cells are co-transfected with these fusion constructs and exposed to biotin, resulting in a BirA-E3 ligase-catalyzed biotinylation of (-2) AP-Ub when in complex with E2. As the next step, the biotinylated (-2) AP-Ub is transferred covalently to the substrate lysine, which enables an enrichment via denaturing streptavidin pulldown. Substrate candidates can then be identified via mass spectrometry (MS). Our ubiquitin-specific proximity-dependent labeling (Ub-POD) method allows robust biotinylation of the ubiquitylation substrates of a candidate E3 ligase thanks to the wild-type BirA and biotin acceptor peptide fused to the E3 and Ub, respectively. Because of the highly Ub-specific labeling, Ub-POD is more appropriate for identifying ubiquitination substrates compared to other conventional proximity labeling or immunoprecipitation (IP) approaches.

N⁶-methyladenosine (m6A) is an abundant internal mRNA modification with roles in regulating cellular and organismal physiology, including development, differentiation, and disease. The deposition of m6A is highly regulated, with various m6A levels across different environmental conditions, cellular states, and cell types. Available methods for measuring bulk m6A levels are often time-consuming, have low throughput, and/or require specialized instrumentation or data analyses. Here, we present a detailed protocol for measuring bulk m6A levels in purified poly(A) RNA samples with m6A-ELISA using a standard-based approach. Critical steps of the protocol are highlighted and optimized, including poly(A) RNA quality controls and antibody specificity testing. The protocol is fast, scalable, adaptable, and cost-effective. It does not require specialized instrumentation, training, or skills in data analysis. We have successfully tested this protocol on mRNAs isolated from budding yeast and mouse cell lines.

It has been discovered that many phytopathogenic fungi can absorb exogenous double-stranded RNAs (dsRNAs) to silence target genes, inhibiting fungal growth and pathogenicity for plant protection. In our recent report, the beneficial arbuscular mycorrhizal (AM) fungi are capable of acquiring external naked dsRNAs; however, whether the dsRNAs can be delivered into AM fungi through nanocarriers remains to be investigated. Here, we introduce a simple and advanced method for in vitro synthesizing chitosan (CS)/dsRNA polyplex nanoparticles (PNs) to silence the target gene in the AM fungus Rhizophagus irregularis. This method is straightforward, requiring minimal modifications, and is both efficient and eco-friendly, offering potential for rapid application in elucidating gene functions in AM fungi.

In many plant species, self-incompatibility (SI) is a mechanism that inhibits inbreeding. SI is gametophytic in the Solanaceae, with specificity determined by S-ribonucleases (S-RNases) in the pistil and S-locus F-box proteins (SLFs) in the pollen. The role of these proteins has been studied extensively in the Solanaceae, often using Petunia as a model. Using degenerate PCR and Sanger sequencing, this protocol identified three SLF sequences from self-incompatible diploid potato (Solanum okadae). While SLFs are well-characterized in model species like Petunia, there is limited sequence data and no standardized protocols for identifying SLFs in non-model species such as S. okadae, hindering broader insights into SI across the Solanaceae. This protocol fills that gap by using degenerate PCR and Sanger sequencing with primers designed from conserved Petunia SLF regions to identify SLF sequences in S. okadae. SLF sequences from 10 distinct Solanaceae members sharing maximum identity with the S2-haplotype of Petunia were used to design two pairs of primers targeting different regions of the target sequence. PCR amplification using designed degenerate primers yielded amplicons that were directly sequenced and joined together to get the partial SLF sequence. It was observed that the S. okadae shared an orthologous relation with the Petunia SLF according to the phylogenetic analysis. These SLFs could be used in future SI breakdown experiments via the competitive interaction route. This protocol, including the primer design, is novel for detecting SLF sequences in S. okadae.

We have observed that some proinsulin molecules in pancreatic islets and beta cell lines have incomplete or improper intramolecular disulfide bonds. These misfolded monomers can form intermolecular disulfide-linked complexes. Accurately quantifying the fraction of proinsulin molecules contained in these complexes is challenging. By proinsulin immunoblotting after nonreducing SDS-PAGE, the signal for disulfide-linked complexes can exceed the total proinsulin signal detected after reducing SDS-PAGE (i.e., overestimating the abundance of misfolded species due to antibody affinity differences). However, after modification of the SDS-PAGE and electrotransfer protocol, we have been able to more accurately estimate the fraction of proinsulin monomers folded to the native state, as well as misfolded proinsulin monomers and disulfide-linked complexes. Our improved technique offers the ability to obtain a more precise assessment of proinsulin misfolding under different environmental conditions in beta cells and normal islets and in diabetes.

Inteins are elements translated within host proteins and removed via a unique protein splicing reaction. In this process, the two peptide bonds flanking the intein are rearranged, releasing the intein and leaving a standard peptide bond in its place. Due to their ability to shuffle peptide bonds in a specific and controlled manner, inteins have proven valuable in protein engineering, leading to the development of numerous impactful technologies. In one application, intein-based biosensors link the activity of a host protein to intein excision. Recently, we developed a biosensor to measure protein stability in vivo, in which the removal of an intein-protein fusion is required for antibiotic resistance. In our protocol, cells expressing our biosensor are logarithmically diluted and spotted on agar plates containing increasing levels of antibiotics. Following incubation, quantitative survival curves can be generated. We also developed a dual protein stability sensor where both antibiotic resistance and fluorescence can be used as readouts and demonstrated that co-expression of the chaperonin GroEL can promote survival and fluorescence. Taken together, our novel intein-based biosensor adds to the available tools to measure protein stability within the cellular environment.

Reverse genetics systems in virology are technologies used to generate recombinant viruses, enabling the manipulation of viral genes. Recombinant viruses facilitate the investigation of pathogenesis and the development of antivirals. In studies of positive-sense single-stranded RNA (ssRNA) viruses, a reverse genetics approach typically uses infectious viral cDNA clones derived from bacterial artificial chromosomes and plasmids or from the in vitro ligation of viral cDNA fragments. However, these methods are time-consuming, involve complex procedures, and do not always successfully generate recombinant viruses. Possible reasons for unsuccessful outcomes include i) viral sequences exhibiting toxicity in bacterial systems, ii) the duplication of viral genes observed in some strains, complicating the acquisition of correct cDNA clones, and iii) certain cell lines being highly susceptible to infection but difficult to transfect with nucleotides. For these reasons, a simple and rapid reverse genetics system is needed to accelerate research on ssRNA viruses. The circular polymerase extension reaction (CPER) method offers a solution by eliminating the need for molecular cloning in bacteria, enabling the generation of recombinant viruses over a shorter timeframe. This method has been widely adopted for the study of ssRNA viruses, including SARS-CoV-2 and flaviviruses. Recently, we expanded the CPER method for ssRNA viruses using internal ribosome entry site (IRES)-mediated translation. This protocol details the experimental procedures, using bovine viral diarrhea virus as an example—one of the most challenging viruses for generating viral cDNA clones because of the factors listed above.

In molecular diagnosis, DNA extraction kits are sample-specific and proprietary, preventing lateral distribution among similar facilities from different sectors to alleviate supply shortages during a crisis. Previous fast extraction protocols such as detergent-based ones allow fast DNA extraction for nucleic acid amplification tests (NAAT), mainly polymerase chain reaction (PCR). The use of NaOH (dense alkali) to rupture cells and nuclei and destabilize the conformation of DNases might alleviate shortages and costs while retaining enough robustness to treat complicated samples with minimal environmental and logistical footprint. Biological samples are hand-crushed using a pestle in 1.5 mL tubes with 360 μL of 0.2 M NaOH for 3–5 min and incubated at 75 °C for 10 min. For immediate use, 115.2 μL of 1 M Tris (pH 8) and 364.8 μL nuclease-free water are added, and the sample is vortexed for 10 s and spun at 10,000× g for 3 min; then, 700 μL is transferred to a clean microtube. Two serial dilutions follow, and all concentrations are used as templates for PCR. A refined, storable extract can be produced by adding 70 μL of HCl 1 M (instead of Tris-HCl) and one volume of cold isopropanol to the extract for standard precipitation. This method can increase throughput in emergencies by field deployment in resource-limited settings (RLS) or allow benchtop backup in cases of acquisition disruption or sample surge in established facilities. The crude extract can be used for immediate PCR in both benchtop and portable thermocyclers, thus allowing NAAT in resource-limited settings with low costs and waste footprint or during prolonged crises, where supply chain failures may occur. The refined version produces alcohol-precipitated nucleic acids, suitable for both immediate use and for storage or dispatch for spatiotemporally separate analysis while offering much better amplification quality with a small increase in time and minimal increase in expendables/chemicals needed.