Tissue-Specific RNA Localization in the Uterus During Implantation, Decidualization and Placentation: Technical Nuances of Various Labeling Approaches.

In situ hybridization (ISH) is a sensitive method used to localize a specific sequence of DNA or RNA in biological samples, including cells, tissue sections or whole organs. RNA ISH can be used to determine spatial gene expression using a single-stranded probe with a reverse-complementary sequence. Cell-specific gene expression has been studied using mRNA and protein levels. Signals produced by RNA probes are usually more specific than those produced by antibodies in immunostaining. Currently, ISH is the most widely used method to localize mRNA molecules. Traditionally, probes were labeled with radioactive isotopes, but the cumbersome procedures and potential health risk limit their acceptance. Recently, probes labeled with nonradioactive materials including digoxigenin, biotin and various fluorophores have been developed. The tyramide signal amplification system further enhances the sensitivity of detection. These methods have been applied in numerous studies in various tissues including reproductive organs. This article details three methods of RNA in situ hybridization: radioactive in situ hybridization, digoxigenin in situ hybridization, and digoxigenin-tyramide signal amplification fluorescein in situ hybridization. The pros and cons of each protocol are discussed. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Radioactive in situ hybridization (radioactive-ISH) Basic Protocol 2: Digoxigenin in situ hybridization (DIG-ISH) Basic Protocol 3: Digoxigenin-tyramide signal amplification fluorescein in situ hybridization (DIG-TSA-FISH).

Digoxigenin-labeled RNA probes for untranslated regions enable the isoform-specific gene expression analysis of myosin heavy chains in whole-mount in situ hybridization.

Myosin heavy chains (MyHCs), which are encoded by myosin heavy chain (Myh) genes, are the most abundant proteins in myofiber. Among the 11 sarcomeric Myh isoform genes in the mammalian genome, seven are mainly expressed in skeletal muscle. Myh genes/MyHC proteins share a common role as force producing units with highly conserved sequences, but have distinct spatio-temporal expression patterns. As such, the expression patterns of Myh genes/MyHC proteins are considered as molecular signatures of specific fiber types or the regenerative status of mammalian skeletal muscles. Immunohistochemistry is widely used for identifying MyHC expression patterns; however, this method is costly and is not ideal for whole-mount samples, such as embryos. In situ hybridization (ISH) is another versatile method for the analysis of gene expression, but is not commonly applied for Myh genes, partly because of the highly homologous sequences of Myh genes. Here we demonstrate that an ISH analysis with the untranslated region (UTR) sequence of Myh genes is cost-effective and specific method for analyzing the Myh gene expression in whole-mount samples. Digoxigenin (DIG)-labeled antisense probes for UTR sequences, but not for protein coding sequences, specifically detected the expression patterns of respective Myh isoform genes in both embryo and adult skeletal muscle tissues. UTR probes also revealed the isoform gene-specific polarized localization of Myh mRNAs in embryonic myofibers, which implied a novel mRNA distribution mechanism. Our data suggested that the DIG-labeled UTR probe is a cost-effective and versatile method to specifically detect skeletal muscle Myh genes in a whole-mount analysis.

Ocimum sanctum, OscWRKY1, regulates phenylpropanoid pathway genes and promotes resistance to pathogen infection in Arabidopsis.

KEY MESSAGE: OscWRKY1 from Ocimum sanctum positively regulates phenylpropanoid pathway genes and rosmarinic acid content. OscWRKY1 overexpression promotes resistance against bacterial pathogen in Arabidopsis. WRKY transcription factor (TF) family regulates various developmental and physiological functions in plants. PAL genes encode enzymes which are involved in plant defense responses, but the direct regulation of PAL genes and phenylpropanoid pathway through WRKY TF's is not well characterized. In the present study, we have characterized an OscWRKY1 gene from Ocimum sanctum which shows induced expression by methyl jasmonate (MeJA), salicylic acid (SA), and wounding. The recombinant OscWRKY1 protein binds to the DIG-labeled (Digoxigenin) W-box cis-element TTGAC[C/T] and activates the LacZ reporter gene in yeast. Overexpression of OscWRKY1 enhances Arabidopsis resistance towards Pseudomonas syringae pv. tomato Pst DC3000. Upstream activator sequences of PAL and C4H have been identified to contain the conserved W-box cis-element (TTGACC) in both O. sanctum and Arabidopsis. OscWRKY1 was found to interact with W-box cis-element present in the PAL and C4H promoters. Silencing of OscWRKY1 using VIGS resulted in reduced expression of PAL, C4H, COMT, F5H and 4CL transcripts. OscWRKY1 silenced plants exhibit reduced PAL activity, whereas, the overexpression lines of OscWRKY1 in Arabidopsis exhibit increased PAL activity. Furthermore, the metabolite analysis of OscWRKY1 silenced plants showed reduced rosmarinic acid content. These results revealed that OscWRKY1 positively regulates the phenylpropanoid pathway genes leading to the alteration of rosmarinic acid content and enhances the resistance against bacterial pathogen in Arabidopsis.

Analysis of Spatial Gene Expression at the Cellular Level in Stony Corals.

Scleractinians, or stony corals, are colonial animals that possess a high regenerative capacity and a highly diverse innate immune system. As such they present the opportunity to investigate the interconnection between regeneration and immunity in a colonial animal. Understanding the relationship between regeneration and immunity in stony corals is of further interest as it has major implications for coral reef health. One method for understanding the role of innate immunity in scleractinian regeneration is in situ hybridization using RNA probes. Here we describe a protocol for in situ hybridization in adult stony corals using a digoxigenin (DIG)-labeled RNA antisense probe which can be utilized to investigate the spatial expression of immune factors during regeneration.

Sensitive Fluorescence In Situ Hybridization on Semithin Sections of Adult Schistosoma mansoni Using DIG-Labeled RNA Probes.

In situ hybridization is a tool for evaluation of gene expression within tissues or single cells. This protocol describes optimized sensitive fluorescence detection of gene transcripts (mRNAs) in semithin sections of Schistosoma mansoni adult worms using specifically designed and labeled RNA probes. Due to improved methodologies in tissue preservation, sectioning, amplification of fluorescent signal, and prehybridization tissue treatment, it is possible to detect transcripts in the fine structures of schistosomes. The protocol is sensitive enough to detect very low abundance targets. This procedure is optimized for tissues derived from S. mansoni adult worms; however, it can be successfully applied to other trematode species.

Wound Healing, Inflammation, and Corneal Ultrastructure After SMILE and Femtosecond Laser-Assisted LASIK: A Human Ex Vivo Study.

PURPOSE: To assess the wound healing, inflammation, and tissue ultrastructure in the human corneal stroma after small incision lenticule extraction (SMILE) and femtosecond laser-assisted LASIK (FS-LASIK). METHODS: Sixteen corneoscleral discs of 16 human donors unsuitable for corneal transplantation were obtained from an eye bank. Eight eyes underwent SMILE with -5.00 diopters (D) of myopic correction; in 3 of them the lenticule was not extracted. Further 5 donor corneas were subjected to FS-LASIK with -5.00 D ablation, and 3 eyes served as the control group without surgical intervention. Postoperatively, specimens were incubated in organ culture medium for 72 hours before being subjected to immunofluorescence staining for CD11b, Ki67, fibronectin, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling assay, and high-magnification scanning electron microscopy. RESULTS: Keratocyte apoptosis, keratocyte proliferation, and infiltration of immune cells were generally mild and comparable between FS-LASIK and SMILE (irrespective of surgical lenticule extraction). By staining for fibronectin, we observed a trend toward milder fibrotic response in the corneal stroma after SMILE than after FS-LASIK. On the contrary, scanning electron microscopy analysis revealed a smoother, more regular ultrastructural appearance of the residual corneal bed after FS-LASIK. CONCLUSIONS: Corneal stromal wound healing after SMILE and FS-LASIK was virtually identical with respect to keratocyte proliferation and apoptosis in the human donor eye model. Although reactive fibrosis adjacent to the laser application site appeared less marked after SMILE, the stromal bed after LASIK exhibited a smoother surface texture. [J Refract Surg. 2018;34(6):393-399.].

CSAR Benchmark Exercise 2013: Evaluation of Results from a Combined Computational Protein Design, Docking, and Scoring/Ranking Challenge.

Community Structure-Activity Resource (CSAR) conducted a benchmark exercise to evaluate the current computational methods for protein design, ligand docking, and scoring/ranking. The exercise consisted of three phases. The first phase required the participants to identify and rank order which designed sequences were able to bind the small molecule digoxigenin. The second phase challenged the community to select a near-native pose of digoxigenin from a set of decoy poses for two of the designed proteins. The third phase investigated the ability of current methods to rank/score the binding affinity of 10 related steroids to one of the designed proteins (pKd = 4.1 to 6.7). We found that 11 of 13 groups were able to correctly select the sequence that bound digoxigenin, with most groups providing the correct three-dimensional structure for the backbone of the protein as well as all atoms of the active-site residues. Eleven of the 14 groups were able to select the appropriate pose from a set of plausible decoy poses. The ability to predict absolute binding affinities is still a difficult task, as 8 of 14 groups were able to correlate scores to affinity (Pearson-r > 0.7) of the designed protein for congeneric steroids and only 5 of 14 groups were able to correlate the ranks of the 10 related ligands (Spearman-ρ > 0.7).

The selection performance of an antibody library displayed on filamentous phage coat proteins p9, p3 and truncated p3.

BACKGROUND: Filamentous phage display has become an ordinary tool to engineer antibody fragments. Several capsid proteins have been applied for displaying antibodies, of which gene III (p3) protein is used the most followed by experiments with gene IX (p9) protein. Despite the popularity, there are no library scale studies to objectively compare differences in the selection performance of the libraries, when displayed via different capsid proteins. RESULTS: In this study, an identical antibody repertoire was displayed as Fab fragments on p9, p3 and truncated p3 (p3Δ). In addition, the library clones were displayed as ScFv fragments on p3Δ and the Fab-p3 display valency was modulated by hyperphage and VCS-M13 superinfections. The selection performances of the libraries were followed in repeated parallel panning reactions against streptavidin (STR) and digoxigenin (DIG). Selection was successful with all display formats, but the enrichment of specific clones from Fab-p9 library was clearly less efficient than from the other libraries. The most diverse outputs were obtained from p3Δ display and the highest affinity anti-DIG antibodies from the ScFv repertoire. Unfortunately, the number of retrieved specific clones was too low for explicit analysis of the differences in the number of obtained unique clones from each library. However, severe reduction in sequence diversity was observed in p3-Fab libraries prior to panning, which in turn, materialized as a low number of unique specific clones. Oligovalent display by hyperphage resulted in a higher number of unique clones, but the same highest affinity anti-DIG Fab was recovered also by VCS-M13 superinfection. CONCLUSIONS: The compromised enrichment of the target-specific clones from the Fab repertoire as a fusion to p9 capsid protein in our experiments, the significant loss of functional diversity in Fab-p3 library after single phage packing cycle and the retrieval of higher affinity anti-digoxigenin clones as ScFv molecules than as Fab molecules from the same source repertoire indicate that the chosen display format may have a significant impact on the selection outcome. This study demonstrates that in addition to library content, also display related issues, should be taken into consideration when planning directed evolution experiments.

Tethered particle analysis of supercoiled circular DNA using peptide nucleic acid handles.

This protocol describes how to monitor individual naturally supercoiled circular DNA plasmids bound via peptide nucleic acid (PNA) handles between a bead and a surface. The protocol was developed for single-molecule investigation of the dynamics of supercoiled DNA, and it allows the investigation of both the dynamics of the molecule itself and of its interactions with a regulatory protein. Two bis-PNA clamps designed to bind with extremely high affinity to predetermined homopurine sequence sites in supercoiled DNA are prepared: one conjugated with digoxigenin for attachment to an anti-digoxigenin-coated glass cover slide, and one conjugated with biotin for attachment to a submicron-sized streptavidin-coated polystyrene bead. Plasmids are constructed, purified and incubated with the PNA handles. The dynamics of the construct is analyzed by tracking the tethered bead using video microscopy: less supercoiling results in more movement, and more supercoiling results in less movement. In contrast to other single-molecule methodologies, the current methodology allows for studying DNA in its naturally supercoiled state with constant linking number and constant writhe. The protocol has potential for use in studying the influence of supercoils on the dynamics of DNA and its associated proteins, e.g., topoisomerase. The procedure takes ~4 weeks.

In situ hybridization (both radioactive and nonradioactive) and spatiotemporal gene expression analysis.

Section in situ hybridization using either radioactive or nonradioactive labeled cDNA probes is an invaluable technique that enables the investigator to detect and localize mRNA expression within tissue sections and cells. Here, we describe the labeling of (35)S-UTP radioactive and nonradioactive digoxigenin probes, preparation of tissue sections, hybridization, and washing of non-hybridized probes, followed by the detection of radioactive signals via dipping in nuclear emulsion and the immunohistochemical and subsequent colorimetric detection of nonradioactive signals.

Simple and nonradioactive detection of microRNAs using digoxigenin (DIG)-labeled probes with high sensitivity.

The discovery of microRNAs (miRNAs), which are ∼21-23 nucleotides that can regulate targeted mRNA by transcript cleavage or protein translation suppression, has changed the landscape of biomedical field greatly. At present, Northern blot analysis based on radioisotopes is still the most popular method on the detection of miRNAs for its high sensitivity. However, radioisotopes have been known for certain disadvantages, such as instability, expense, and safety; thus, developing a nonradioactive and highly sensitive method is needed. Here, we report a simple, nonradioactive, and sensitive method for miRNAs detection based on 5'-phos-3'-DIG-labeled probes prepared through splinted ligation and EDC cross-linking (DSLE). The method was more sensitive than traditional Northern blots with a DIG-labeled DNA probe and can detect as low as 2 fmol of miRNAs. The whole procedure can be completed within 6-8 h. DSLE method is very convenient, cost-effective, time-saving, and highly sensitive.

Analysis of DNA damage and repair by comet fluorescence in situ hybridization (Comet-FISH).

A useful tool in the detection of overall and region-specific DNA damage is the Comet-FISH technique. This method combines two well-established methods, the Comet assay (single cell gel electrophoresis), which makes it possible to detect and quantify DNA damage at the single cell level, and FISH (fluorescence in situ hybridization), a technique that allows the specific detection of selected DNA sequences. The influence of specific substances such as water pollutants or food ingredients on individual cells can be measured with the alkaline version of the Comet assay, which involves the embedding of cells in agarose on microscopic slides, lysis of cells, and separation of DNA via electrophoresis. In damaged cells a "comet tail" is formed by fractured DNA migrating from the nucleus (head of the comet) in the electric field.The damaged DNA (DNA strand breaks) correlates with the percentage of DNA in the tail. In combination with the FISH method, DNA damage or repair capacity in single cells can be measured using labelled probes, which hybridize to specific DNA sequences of interest. This protocol exemplarily provides a description of the Comet-FISH technique for the detection of DNA damage using hydrogen peroxide as a genotoxic model substance.

Immunohistochemistry and RNA in situ hybridization in mouse brain development.

During development, the mouse brain is progressively divided into functionally distinct compartments. Numerous neuronal and glial cell types are subsequently generated in response to various inductive signals. Each cell expresses a unique combination of genes encoding proteins from transcription factors to neurotransmitters that define its role in brain function. To understand these important and highly sophisticated processes, it is critical to accurately locate the various proteins and cells that produce them. In this chapter, we introduce the techniques of immunohistochemistry, which detects the localization of specific proteins, and RNA in situ hybridization, which enables the visualization of specific mRNAs.

Trimeric autotransporter DsrA is a major mediator of fibrinogen binding in Haemophilus ducreyi.

Haemophilus ducreyi is the etiologic agent of the sexually transmitted genital ulcer disease chancroid. In both natural and experimental chancroid, H. ducreyi colocalizes with fibrin at the base of the ulcer. Fibrin is obtained by cleavage of the serum glycoprotein fibrinogen (Fg) by thrombin to initiate formation of the blood clot. Fg binding proteins are critical virulence factors in medically important Gram-positive bacteria. H. ducreyi has previously been shown to bind Fg in an agglutination assay, and the H. ducreyi Fg binding protein FgbA was identified in ligand blotting with denatured proteins. To better characterize the interaction of H. ducreyi with Fg, we examined Fg binding to intact, viable H. ducreyi bacteria and identified a novel Fg binding protein. H. ducreyi bound unlabeled Fg in a dose-dependent manner, as measured by two different methods. In ligand blotting with total denatured cellular proteins, digoxigenin (DIG)-Fg bound only two H. ducreyi proteins, the trimeric autotransporter DsrA and the lectin DltA; however, only the isogenic dsrA mutant had significantly less cell-associated Fg than parental strains in Fg binding assays with intact bacteria. Furthermore, expression of DsrA, but not DltA or an empty vector, rendered the non-Fg-binding H. influenzae strain Rd capable of binding Fg. A 13-amino-acid sequence in the C-terminal section of the passenger domain of DsrA appears to be involved in Fg binding by H. ducreyi. Taken together, these data suggest that the trimeric autotransporter DsrA is a major determinant of Fg binding at the surface of H. ducreyi.

Computational design of ligand-binding proteins with high affinity and selectivity.

The ability to design proteins with high affinity and selectivity for any given small molecule is a rigorous test of our understanding of the physiochemical principles that govern molecular recognition. Attempts to rationally design ligand-binding proteins have met with little success, however, and the computational design of protein-small-molecule interfaces remains an unsolved problem. Current approaches for designing ligand-binding proteins for medical and biotechnological uses rely on raising antibodies against a target antigen in immunized animals and/or performing laboratory-directed evolution of proteins with an existing low affinity for the desired ligand, neither of which allows complete control over the interactions involved in binding. Here we describe a general computational method for designing pre-organized and shape complementary small-molecule-binding sites, and use it to generate protein binders to the steroid digoxigenin (DIG). Of seventeen experimentally characterized designs, two bind DIG; the model of the higher affinity binder has the most energetically favourable and pre-organized interface in the design set. A comprehensive binding-fitness landscape of this design, generated by library selections and deep sequencing, was used to optimize its binding affinity to a picomolar level, and X-ray co-crystal structures of two variants show atomic-level agreement with the corresponding computational models. The optimized binder is selective for DIG over the related steroids digitoxigenin, progesterone and ß-oestradiol, and this steroid binding preference can be reprogrammed by manipulation of explicitly designed hydrogen-bonding interactions. The computational design method presented here should enable the development of a new generation of biosensors, therapeutics and diagnostics.

Novel multiplexed assay for identifying SH2 domain antagonists of STAT family proteins.

Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide variety of human tumors. The activity of STAT depends on their Src homology 2 (SH2) domain-mediated binding to sequences containing phosphorylated tyrosine. Thus, antagonizing this binding is a feasible approach to inhibiting STAT activation. We have developed a novel multiplexed assay for STAT3- and STAT5b-SH2 binding, based on amplified luminescent proximity homogeneous assay (Alpha) technology. AlphaLISA and AlphaScreen beads were combined in a single-well assay, which allowed the binding of STAT3- and STAT5b-SH2 to phosphotyrosine peptides to be simultaneously monitored. Biotin-labeled recombinant human STAT proteins were obtained as N- and C-terminal deletion mutants. The spacer length of the DIG-labeled peptide, the reaction time, and the concentration of sodium chloride were optimized to establish a HTS system with Z' values of greater than 0.6 for both STAT3- and STAT5b-SH2 binding. We performed a HTS campaign for chemical libraries using this multiplexed assay and identified hit compounds. A 2-chloro-1,4-naphthalenedione derivative, Compound 1, preferentially inhibited STAT3-SH2 binding in vitro, and the nuclear translocation of STAT3 in HeLa cells. Initial structure activity relationship (SAR) studies using the multiplexed assay showed the 3-substituent effect on both the activity and selectivity of STAT3 and STAT5b inhibition. Therefore, this multiplexed assay is useful for not only searching for potential lead compounds but also obtaining SAR data for developing new STAT3/STAT5b inhibitors.

General introduction to in situ hybridization protocol using nonradioactively labeled probes to detect mRNAs on tissue sections.

In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA or RNA strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue (In Situ) or in the entire tissue (whole mount ISH). Localization of endogenous transcripts is a desirable approach for confirming expression patterns. This is distinct from immunohistochemistry, which usually localizes proteins in tissue sections. DNA ISH can be used to determine the structure of chromosomes. However, RNA ISH (hybridization histochemistry) is used to measure and localize mRNAs and other transcripts within tissue sections or whole mounts. RNA-RNA hybrids approach may offer increased sensitivity, which is more stable than that of DNA-RNA hybrids. Here we describe the efficient ISH protocol for nonradioactive (i.e., in direct methods using digoxigenin (DIG) system) RNA probes, and it can be performed in less than 3 days.