Antisense Probes Research Articles

The distribution of Hypocretin/Orexin receptor mRNA in the mouse and human brain

Hypocretin (Hcrtr, HCRTR) / orexin (OX) receptors modulate a range of neurobiological functions and are drug targets for several disorders. Mapping the distribution of receptors in the brain can inform their function and guide targeting of specific disorders. Although studied in rodents, orexin receptor distribution has remained relatively unexplored in humans, and thus there is also a paucity of comparative anatomy. The aim of this study was therefore to map the distribution of hypocretin/orexin receptor mRNA in selected regions of the mouse and human brain by non-radioactive in situ hybridization (ISH) using digoxigenin (DIG)-labelled cRNA anti-sense probes. Data revealed both distinct and overlapping patterns of distributions of Hcrtr1/HCRTR1 and Hcrtr2/HCRTR2 mRNA suggesting that the functions of the orexin system are mediated differently by each receptor. In the mouse brain, the highest expression of Hcrtr1 mRNA was in the locus coeruleus (LC) whereas Hcrtr2 mRNA was most abundant in the lateral hypothalamus (LH). The human caudate nuclei showed significant expression of both HCRTR1 and HCRTR2 mRNA, whereas the mouse predominantly expressed Hcrtr2 mRNA. The noradrenergic neurons of the human LC showed high signals for both HCRTR1 (71.7%) and HCRTR2 (81.5%) mRNA. Expression of HCRTR2 mRNA in non-noradrenergic human LC cells was also notable. The distribution pattern in mouse and human brains is consistent with the involvement of the orexin system in arousal and the sleep/wake cycle in both species, however, variations in receptor subtype expression profiles suggests that species differences in responses to orexin receptor ligands may be expected.

Chemi-Northern: a versatile chemiluminescent northern blot method for analysis and quantitation of RNA molecules.

This report describes a chemiluminescence-based detection method for RNAs on northern blots, designated Chemi-Northern. This approach builds on the simplicity and versatility of northern blotting, while dispensing of the need for expensive and cumbersome radioactivity. RNAs are first separated by denaturing gel electrophoresis, transferred to a nylon membrane, and then hybridized to a biotinylated RNA or DNA antisense probe. Streptavidin conjugated with horseradish peroxidase and enhanced chemiluminescence substrate are then used to detect the probe bound to the target RNA. Our results demonstrate the versatility of this method in detecting natural and engineered RNAs expressed in cells, including messenger and noncoding RNAs. We show that Chemi-Northern detection is sensitive and fast, detecting attomole amounts of RNA in as little as 1 sec, with high signal intensity and low background. The dynamic response displays excellent linearity. Using Chemi-Northern, we measure the reproducible, statistically significant reduction of mRNA levels by human sequence-specific RNA-binding proteins, PUM1 and PUM2. Additionally, we measure the interaction of the poly(A) binding protein, PABPC1, with polyadenylated mRNA. Thus, the Chemi-Northern method provides a versatile, simple, and cost-effective method to enable researchers to analyze expression, processing, binding, and decay of RNAs.

N-3 Polyunsaturated Fatty Acid (N-3 PUFA) Derived Lipid Mediators Increase Hematopoiesis during Zebrafish Embryogenesis

Polyunsaturated fatty acids (PUFAs), which include n-3 PUFAs and n-6 PUFAs, are essential fatty acids that we can only get from diet. The richest source of PUFAs is fish oil. N-3 PUFAs have been used in the prevention and treatment of coronary artery disease, diabetes, hypertension, arthritis, cancer, and other inflammatory and autoimmune disorders in humans. Lipid mediators derived from n-3 PUFAs are implicated in mediating the resolution of inflammation. However, their effect on hematopoietic stem and progenitor cell (HSPC) proliferation and differentiation and on hematopoiesis has not been studied. Therefore, in this project, we investigated the pro-hematopoietic effects of n-3 PUFA-derived lipid mediators. We exposed wild-type zebrafish embryos to different n-3 PUFA-derived lipid mediators. After fixation, we performed whole mount in situ hybridization (WISH) using antisense RNA probes such as c-myb (hematopoietic stem cell marker), gata1 (erythroid progenitor cell marker), hbbe1 (erythrocyte marker), l-plastin (macrophage and neutrophil cell marker ), mpx (neutrophil cell marker), lyz (neutrophil cell marker), and rag1 (immature lymphocyte marker); reverse transcription-quantitative polymerase chain reaction (RT-qPCR); and fluorescence-activated cell sorting (FACS) to understand the effect of lipid mediator exposure on the proliferation of HSPCs, erythroid progenitor cells, erythrocytes, granulocytic cells, neutrophils, macrophages, and T lymphocytes in different development stages during vertebrate hematopoiesis. In addition, we also used o-dianisidine staining to study red blood cell (RBC) formation and transgenic zebrafish to observe different blood lineages including HSPCs, neutrophils, macrophages, erythroid progenitors, and erythrocytes with fluorescent microscopy. We identified two novel lipid mediators which promote HSPC proliferation, enhance erythroid progenitor cell and erythrocyte production, and increase neutrophil cell number. These two molecules could be applied as novel therapeutics for blood disorders and blood cancers such as anemia and leukemia in humans. These two compounds can be intensely investigated to become candidate supplements to enhance HSPC amplification before HSPC transplantation for leukemia patients.

Presenting technological workflows

Presenting technological workflows

Fluorescent In Situ Hybridization in Embryos of the African Turquoise Killifish Nothobranchius furzeri.

Precisely where and when a given gene is expressed is crucial for our understanding of developmental and cell biology but determining this is often constrained by detection limits. Here, we describe a technique for visualization of low-copy mRNA in Nothobranchius furzeri embryos using tyramide signal amplification (TSA). In this protocol, an anti-sense digoxigenin-labeled RNA probe is hybridized to mRNA in situ. Anti-digoxigenin antibodies conjugated to horseradish peroxidase (POD) are then bound to the probe and reacted with fluorescently labeled tyramide. Combining this method with a counterstain, such as DAPI, allows for the detection of mRNA at a single-cell resolution.

Experience-dependent changes in affective valence of taste in male mice

Taste plays an essential role in the evaluation of food quality by detecting potential harm and benefit in what animals are about to eat and drink. While the affective valence of taste signals is supposed to be innately determined, taste preference can also be drastically modified by previous taste experiences of the animals. However, how the experience-dependent taste preference is developed and the neuronal mechanisms involved in this process are poorly understood. Here, we investigate the effects of prolonged exposure to umami and bitter tastants on taste preference using two-bottle tests in male mice. Prolonged umami exposure significantly enhanced umami preference with no changes in bitter preference, while prolonged bitter exposure significantly decreased bitter avoidance with no changes in umami preference. Because the central amygdala (CeA) is postulated as a critical node for the valence processing of sensory information including taste, we examined the responses of cells in the CeA to sweet, umami, and bitter tastants using in vivo calcium imaging. Interestingly, both protein kinase C delta (Prkcd)-positive and Somatostatin (Sst)-positive neurons in the CeA showed an umami response comparable to the bitter response, and no difference in cell type-specific activity patterns to different tastants was observed. Meanwhile, fluorescence in situ hybridization with c-Fos antisense probe revealed that a single umami experience significantly activates the CeA and several other gustatory-related nuclei, and especially CeA Sst-positive neurons were strongly activated. Intriguingly, after prolonged umami experience, umami tastant also significantly activates the CeA neurons, but the Prkcd-positive neurons instead of Sst-positive neurons were highly activated. These results suggest a relationship between amygdala activity and experience-dependent plasticity developed in taste preference and the involvement of the genetically defined neural populations in this process.

Correction to: 'Identification of sex chromosome and sex-determining gene of southern catfish based on XX, XY and YY genome sequencing' (2022) by Zheng et al.

Correction to: 'Identification of sex chromosome and sex-determining gene of southern catfish based on XX, XY and YY genome sequencing' (2022) by Zheng et al.

Electrochemical aptasensor detection of electron transfer flavoprotein subunit beta for leptospirosis diagnosis.

Electron transfer flavoprotein subunit beta (ETFB) of Leptospira interrogans is a biomarker for diagnosing leptospiral infection. Thus, the ETFB-specific nuclease-resistant RNA aptamer ETFB3-63 was developed and used in an electrochemical aptasensor to assay ETFB. Although the majority of reported biosensors detect various genes and antibodies of L. interrogans, this is the first attempt to construct an electrochemical biosensor to detect ETFB protein for the diagnosis of leptospiral infection. The ETFB protein can be detected without any extraction phase. In this assay, a single-stranded DNA probe complementary to the ETFB3-63 sequence was immobilized on a screen-printed carbon electrode (SPCE). The aptamer was then incubated and hybridized with the antisense probe on the SPCE. In the presence of ETFB, the aptamer dissociates from the aptamer/probe complex on the SPCE to bind with the protein. Methylene blue was then added to intercalate with the remaining hybridized aptamers, and its signal was measured using differential pulse voltammetry. The signal arising from the intercalated methylene blue decreased with increasing concentration of ETFB, showing a linear response in the range of 50-500 nM of ETFB and 10 to 109 leptospira cells per mL, respectively. The aptasensor signal was also specific to L. interrogans but not to 12 related bacteria tested. In addition, the aptasensor showed similar performance in detecting ETFB spiked in human serum to that in buffer, indicating that proteins in the serum do not interfere with the assay. Therefore, this assay has great potential to develop into a point-of-care electrochemical device that is accurate, cost-effective, and user-friendly for leptospirosis diagnosis.

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.

Investigation on small molecule-aptamer dissociation equilibria based on antisense displacement probe

Food safety is a major issue to public health and have attracted global attention. Fast, sensitive, and reliable detection methods for food hazardous substances is highly desirable. Aptamers which can bind to the target molecules with high affinity and specificity represent an attractive tool for the recognition of food hazardous substances, which play an important role in the development and application of new food safety detection technology. But current assays for characterizing small molecule-aptamer binding are limited by either the mass sensitivity or the size differentiation ability. Herein, we proposed a comprehensive method for assessing the dissociation equilibria of small molecule-aptamer, which is immobilized-free under ambient conditions. The design employs the Le Chatelier's principle and could be used to effectively measure small molecule-aptamer interactions. ATP binding aptamer and anti-aflatoxin B1 aptamer were used as the model system to determine their affinity, in which their dissociation equilibria measurements are in excellent close to their previous work. Due to the simplicity and sensitivity of this new method, we believe that it could be recommended as an effective tool for characterizing small molecule-aptamer interactions and promote the further application of small molecular aptamer in food safety.

Whole-Mount In Situ Hybridization (WISH) for Salamander Embryos and Larvae.

Whole-mount in situ hybridization (WISH) is widely used to visualize transcribed gene sequences (mRNA) in developing embryos, larvae, and other nucleotide probe permeable tissue samples. This methodology involves the hybridization of an antisense nucleotide probe to the target mRNA, followed by chromogen or fluorescence-based detection. Here we describe a protocol for the spatiotemporal analysis of mRNA transcripts in axolotl embryos/larvae using digoxigenin-labeled riboprobes, anti-digoxigenin alkaline phosphatase, Fab fragments antibody, and NBT/BCIP chromogen detection.

Sensitive and Specific MicroRNA In Situ Hybridization Using Partially Methylated Phosphotriester Antisense DNA Probes

Sensitive and Specific MicroRNA <i>In Situ</i> Hybridization Using Partially Methylated Phosphotriester Antisense DNA Probes

Primordial germ cell identification and traceability during the initial development of the Siluriformes fish Pseudopimelodus mangurus.

Primordial germ cells (PGCs) are responsible for generating all germ cells. Therefore, they are essential targets to be used as a tool for the production of germline chimeras. The labeling and route of PGCs were evaluated during the initial embryonic development of Pseudopimelodus mangurus, using whole-mount in situ hybridization (WISH) and mRNA microinjection in zygotes. A specific antisense RNA probe constituted by a partial coding region from P. mangurus nanos3 mRNA was synthesized for the WISH method. RNA microinjection was performed using the GFP gene reporter regulated by translation regulatory P. mangurus buc and nanos3 3'UTR sequences, germline-specific markers used to describe in vivo migration of PGCs. Nanos3 and buc gene expression was evaluated in tissues for male and female adults and initial development phases and larvae from the first to seventh days post-hatching. The results from the WISH technique indicated the origin of PGCs in P. mangurus from the aggregations of nanos3 mRNA in the cleavage grooves and the signals obtained from nanos3 probes corresponded topographically to the migratory patterns of the PGCs reported for other fish species. Diffuse signals were observed in all blastomeres until the 16-cell stage, which could be related to the two sequences of the nanos3 3'UTR observed in the P. mangurus unfertilized egg transcriptome. Microinjection was not successful using GFP-Dr-nanos1 3'UTR mRNA and GFP-Pm-buc 3'UTR mRNA and allowed the identification of potential PGCs with less than 2% efficiency only and after hatching using GFP-Pm-nanos3 3'UTR. Nanos3 and buc gene expression was reported in the female gonads and from fertilized eggs until the blastula phase. These results provide information about the PGC migration of P. mangurus and the possible use of PGCs for the future generation of germline chimeras to be applied in the conservation efforts of Neotropical Siluriformes species. This study can contribute to establishing genetic banks, manipulating organisms, and assisting in biotechnologies such as transplanting germ cells in fish.

Restriction of SARS-CoV-2 replication in the human placenta

Although SARS-CoV-2 can infect human placental tissue, vertical transmission is rare. Therefore, the placenta may function as a barrier to inhibit viral transmission to the foetus, though the mechanisms remain unclear. In this study, we confirmed the presence of the SARS-CoV-2 genome in human placental tissue by in situ hybridization with antisense probes targeting the spike protein; tissue staining was much lower when using sense probes for the spike protein. To the best of our knowledge, this is the first evidence directly indicating inefficient viral replication in the SARS-CoV-2-infected placenta. Additional studies are required to reveal the detailed mechanisms.

A light-induced small G-protein gem limits the circadian clock phase-shift magnitude by inhibiting voltage-dependent calcium channels.

Calcium signaling is pivotal to the circadian clockwork in the suprachiasmatic nucleus (SCN), particularly in rhythm entrainment to environmental light-dark cycles. Here, we show that a small G-protein Gem, an endogenous inhibitor of high-voltage-activated voltage-dependent calcium channels (VDCCs), is rapidly induced by light in SCN neurons via the calcium (Ca2+)-mediated CREB/CRE transcriptional pathway. Gem attenuates light-induced calcium signaling through its interaction with VDCCs. The phase-shift magnitude of locomotor activity rhythms by light, at night, increases in Gem-deficient (Gem-/-) mice. Similarly, in SCN slices from Gem-/- mice, depolarizing stimuli induce larger phase shifts of clock gene transcription rhythms that are normalized by the application of an L-type VDCC blocker, nifedipine. Voltage-clamp recordings from SCN neurons reveal that Ca2+ currents through L-type channels increase in Gem-/- mice. Our findings suggest that transcriptionally activated Gem feeds back to suppress excessive light-evoked L-type VDCC activation, adjusting the light-induced phase-shift magnitude to an appropriate level in mammals.

Radiosynthesis of a novel antisense imaging probe targeting LncRNA HOTAIR in malignant glioma

BackgroundLong non-coding RNA (LncRNA) HOTAIR was amplified and overexpressed in many human carcinomas, which could serve as a useful target for cancer early detection and treatment. The 99mTc radiolabeled antisense oligonucleotides (ASON) could visualize the expression of HOTAIR and provide a diagnostic value for malignant tumors. The aim of this study was to evaluate whether liposome-coated antisense oligonucleotide probe 99mTc-HYNIC-ASON targeting HOTAIR can be used in in vivo imaging of HOTAIR in malignant glioma xenografts.MethodsThe ASON targeting LncRNA HOTAIR as well as mismatched ASON (ASONM) were designed and modified. The radiolabeling of 99mTc with two probes were via the conjugation of bifunctional chelator HYNIC. Then probes were purified by Sephadex G25 and tested for their radiolabeling efficiency and purity, as well as stability by ITLC (Instant thin-layer chromatography) and gel electrophoresis. Then the radiolabeled probes were transfected with lipofectamine 2000 for cellular uptake test and the next experimental use. Furthermore, biodistribution study and SPECT imaging were performed at different times after liposome-coated 99mTc-HYNIC-ASON/ASONM were intravenously injected in glioma tumor-bearing mice models. All data were analyzed by statistical software.ResultsThe labeling efficiencies of 99mTc-HYNIC-ASON and 99mTc-HYNIC-ASONM measured by ITLC were (91 ± 1.5) % and (90 ± 0.6) %, respectively, and both radiochemical purities were more than 89%. Two probes showed good stability within 12 h. Gel electrophoresis confirmed that the oligomers were successfully radiolabeled no significant degradation were found. Biodistribution study demonstrated that liposome-coated antisense probes were excreted mainly through the kidney and bladder and has higher uptake in the tumor. Meanwhile, the tumor was clearly shown after injection of liposome coated 99mTc-HYNIC-ASON, and its T/M ratio was higher than that in the non-transfection group and mismatched group. No tumor was seen in mismatched and blocking group.ConclusionThe liposome encapsulated 99mTc-HYNIC-ASON probe can be used in the in vivo, real-time imaging of LncRNA HOTAIR expression in malignant glioma.

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.

Characteristic expression of CGRP and osteogenic and vasculogenic markers in the proximal and distal regions of the rib during male mouse development

BackgroundNeuropeptide calcitonin gene-related peptide (CGRP) is a neurotransmitter related to vasculogenesis and osteogenesis during bone formation and organ development. From the foetal period to the postnatal period, the thorax, which is necessary for lung respiration, forms. The thorax exhibits the same cartilage ossification as the bones of the extremities, but a specific system within the thorax exists as costal cartilage after birth. The relationship among CGRP, osteogenesis and vasculogenic markers in the two rib locations during thorax formation is not fully understood. Materials and methodsIn our study, male mice were used to provide ribs under different development conditions on various embryonic days (E12. 5, E14.5, and E17.5) and postnatal days (P1 and P5). The mRNA expression levels of CGRP, vascular endothelial growth factor (VEGF-A), type 1 collagen (Col1a-1), type 2 collagen (Col2a1), neuropeptide Y (NPY), osteocalcin (OCN) and osteopontin (OPN) were analysed by qRT-PCR. We also analysed the mRNA expression of CGRP, VEGF-A and OPN by in situ hybridization. Multivariate modelling with principal component analysis (PCA) was performed to estimate the interactions among the quantitative real-time RT-PCR data. ResultsThe mRNA expression levels of CGRP, VEGF-A, Col2a, Col1a-1, OCN, and NPY in the male mouse rib gradually increased during development. An antisense probe for CGRP mRNA was strongly detected in the central region of the mouse rib at E12.5 and the hypertrophic and ossification zones at E17.5 by in situ hybridization. VEGF-A was also located in the same region as CGRP at E12.5 and E17.5. OPN was strongly detected at the rib formation stage from E14.5 to E17.5. The expression of CGRP also differed between the proximal and distal regions of the rib at E17.5. As demonstrated by in situ hybridization, CGRP continuously participates in cartilage formation in the distal regions of the rib after birth. The PCA revealed that the mRNA expression of CGRP was related to that of Col1a-1 and VEGF-A during rib formation. ConclusionThis study shows that CGRP is involved in vascular and bone formation during rib development and may also be involved in cartilage formation after birth. The findings suggest that CGRP may temporarily participate in bone formation and continuously participate in cartilage formation in the rib, which may also be related to the formation of the anterior thoracic wall after birth

A standardized definition of placental infection by SARS-CoV-2, a consensus statement from the National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health and Human Development SARS-CoV-2 Placental Infection Workshop

Pregnant individuals infected with SARS-CoV-2 have higher rates of intensive care unit admission, oxygen requirement, need for mechanical ventilation, and death than nonpregnant individuals. Increased COVID-19 disease severity may be associated with an increased risk of viremia and placental infection. Maternal SARS-CoV-2 infection is also associated with pregnancy complications such as preeclampsia and preterm birth, which can be either placentally mediated or reflected in the placenta. Maternal viremia followed by placental infection may lead to maternal-fetal transmission (vertical), which affects 1% to 3% of exposed newborns. However, there is no agreed-upon or standard definition of placental infection. The National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health and Human Development convened a group of experts to propose a working definition of placental infection to inform ongoing studies of SARS-CoV-2 during pregnancy. Experts recommended that placental infection be defined using techniques that allow virus detection and localization in placental tissue by one or more of the following methods: in situ hybridization with antisense probe (detects replication) or a sense probe (detects viral messenger RNA) or immunohistochemistry to detect viral nucleocapsid or spike proteins. If the abovementioned methods are not possible, reverse transcription polymerase chain reaction detection or quantification of viral RNA in placental homogenates, or electron microscopy are alternative approaches. A graded classification for the likelihood of placental infection as definitive, probable, possible, and unlikely was proposed. Manuscripts reporting placental infection should describe the sampling method (location and number of samples collected), method of preservation of tissue, and detection technique. Recommendations were made for the handling of the placenta, examination, and sampling and the use of validated reagents and sample protocols (included as appendices).

Identification of RNAs Engaged in Direct RNA-RNA Interaction with a Long Non-Coding RNA

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome by identifying their molecular partners, DNA, proteins and/or RNAs. The latter can interact with lncRNA through networks involving proteins, but they can also be engaged in direct RNA/RNA interactions. We, therefore, developed an easy-to-use RNA pull-down procedure that allowed identification of RNAs engaged in direct RNA/RNA interaction with a lncRNA using psoralen, a molecule that cross-links only RNA/RNA interactions. Bioinformatics modeling of the lncRNA secondary structure allowed the selection of several specific antisense DNA oligonucleotide probes with a strong affinity for regions displaying a low probability of internal base pairing. Since the specific probes that were designed targeted accessible regions throughout the length of the lncRNA, the RNA-interaction zones could be delineated in the sequence of the lncRNA. When coupled with a high throughput RNA sequencing, this protocol can be used for the whole direct RNA interactome studies of a lncRNA of interest.