Production of Digoxigenin-Labeled Riboprobes for In Situ Hybridization Experiments.

Experiments that visualize gene expression in intact tissues or organisms are fundamental to studies of gene function. These experiments, called in situ hybridization, require the production of a riboprobe, which is a labeled antisense RNA corresponding to a particular gene. The most commonly used system for visualizing gene expression via in situ hybridization is the incorporation of a digoxigenin label into an in vitro-transcribed RNA probe. After hybridization of the riboprobe to a target mRNA, its location can be detected via a high-affinity α-digoxigenin antibody conjugated to an alkaline-phosphatase enzyme. The article describes the design and production of digoxigenin-labeled riboprobes transcribed in vitro from template DNA (either plasmid or PCR amplicon). These riboprobes are suitable for use in tissue and whole-mount in situ hybridization protocols. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Plasmid-derived riboprobes Alternate Protocol: PCR-derived riboprobes Basic Protocol 2: Riboprobe synthesis with DIG label.

Production of a bovine IL-12p40 probe and application using in situ hybridization on ruminant fixed tissues.

Pro-inflammatory cytokines (particularly IL-12) are important for initiating protective T helper 1 (Th1)-type immune responses and hence vital for combating intracellular infections and tumours. In situ hybridization (ISH) provides a powerful diagnostic tool allowing the identification and localization of cells producing these mediators in fixed tissues. The objective of this work was to produce a bovine IL-12p40 probe that allows detection of IL-12p40 mRNA in fixed tissues from different ruminant species. The RNA probe sequence is 447bp in length and from a region with high cross-species-sequence homology (>97.3% homology) to the ovine, cervine, caprine and bubaline IL-12p40 genes. ISH was carried out on paraformaldehyde fixed tissues collected from cattle, sheep and goats. The probe was efficient in identifying IL-12p40 expressing cells in fixed tissues from all these species. In conclusion, the IL-12p40 probe was efficient in identifying and localizing cells that express IL-12p40, and provides a good immuno-diagnostic technique to characterize immune responses in fixed tissues.

Embryological manipulations in zebrafish.

Due to the powerful combination of genetic and embryological techniques, the teleost fish Danio rerio has emerged in the last decade as an important model organism for the study of embryonic development. It is relatively easy to inject material such as mRNA or synthetic oligonucleotides to reduce or increase the expression of a gene product. Changes in gene expression can be analyzed at the level of mRNA, by whole-mount in situ hybridization, or at the level of protein, by immunofluorescence. It is also possible to quantitatively analyze protein levels by Western and immunoprecipitation. Cell behavior can be analyzed in detail by cell transplantation and by fate mapping. Because a large number of mutations have been identified in recent years, these methods can be applied in a variety of contexts to provide a deep understanding of gene function that is often more difficult to achieve in other vertebrate model systems.

Quantitative analysis of microRNA in blood serum with protein-facilitated affinity capillary electrophoresis.

MicroRNAs (miRNAs) are small (∼22 nt) regulatory RNAs that are frequently deregulated in cancer and have shown promise as tissue- and blood-based biomarkers for cancer classification and prognostication. Here we present a protein-facilitated affinity capillary electrophoresis (ProFACE) assay for rapid quantification of miRNA levels in blood serum using single-stranded DNA binding protein (SSB) and double-stranded RNA binding protein (p19) as separation enhancers. The method utilizes either the selective binding of SSB to a single-stranded DNA/RNA probe or the binding of p19 to miRNA-RNA probe duplex. For the detection of ultralow amounts of miRNA without polymerase chain reaction (PCR) amplification in blood samples we apply off-line preconcentration of synthetic miRNA-122 from serum by p19-coated magnetic beads followed by online sample stacking in the ProFACE assay. The detection limit is 0.5 fM or 30 000 miRNA molecules in 1 mL of serum as a potential source of naïve miRNAs.

RNA labeling, conjugation and ligation.

Advances in RNA nanotechnology will depend on the ability to manipulate, probe the structure and engineer the function of RNA with high precision. This article reviews current abilities to incorporate site-specific labels or to conjugate other useful molecules to RNA either directly or indirectly through post-synthetic labeling methodologies that have enabled a broader understanding of RNA structure and function. Readily applicable modifications to RNA can range from isotopic labels and fluorescent or other molecular probes to protein, lipid, glycoside or nucleic acid conjugates that can be introduced using combinations of synthetic chemistry, enzymatic incorporation and various conjugation chemistries. These labels, conjugations and ligations to RNA are quintessential for further investigation and applications of RNA as they enable the visualization, structural elucidation, localization, and biodistribution of modified RNA.

Localization and anchorage of maternal mRNAs to cortical structures of ascidian eggs and embryos using high resolution in situ hybridization.

In several species, axis formation and tissue differentiation are the result of developmental cascades which begin with the localization and translation of key maternal mRNAs in eggs. Localization and anchoring of mRNAs to cortical structures can be observed with high sensitivity and resolution by fluorescent in situ hybridization coupled with labeling of membranes and macromolecular complexes. Oocytes and embryos of ascidians--marine chordates closely related to vertebrates--are ideal models to understand how maternal mRNAs pattern the simple ascidian tadpole. More than three dozen cortically localized maternal mRNAs have been identified in ascidian eggs. They include germ cell markers such as vasa or pem-3 and determinants of axis (pem-1), unequal cleavage (pem-1), and muscle cells (macho-1). High resolution localization of mRNAs, proteins, and lipids in whole eggs and embryos and their cortical fragments shows that maternal mRNA determinants pem-1 and macho-1 are anchored to cortical endoplasmic reticulum and segregate with it into small posterior somatic cells. In contrast, mRNAs such as vasa are associated with granular structures which are inherited by the same somatic cells plus adjacent germ cell precursors. In this chapter, we provide detailed protocols for simultaneous localization of mRNAs and proteins to determine their association with cellular structures in eggs and embryos. Using preparations of isolated cortical fragments with intact membranous structures allows unprecedented high resolution analysis and identification of cellular anchoring sites for key mRNAs. This information is necessary for understanding the mechanisms for localizing mRNAs and partitioning them into daughter cells after cleavage.

High-throughput analysis of gene expression on tissue sections by in situ hybridization.

Genome-scale sequencing projects, high-throughput RNAi screens, systematic gene targeting, and system-biology-based network predictions all depend on a validation of biological significance in order to understand the relevance of a particular finding. Such validation, for the most part, rests on low-throughput technologies. This article provides protocols that, in combination with suitable instrumentation, make possible a semi-automated analysis of gene expression on tissue sections by means of in situ hybridization. Knowledge of gene expression localization has the potential to aid, and thereby accelerate, the validation of gene functions.

Optimized method for the synthesis and purification of adenosine--folic acid conjugates for use as transcription initiators in the preparation of modified RNA.

We present an optimized synthetic strategy for the attachment of molecules to 5'-adenosine monophosphate (AMP), which can then be used to label the 5'-end of RNA by T7 RNA polymerase mediated in vitro transcription. Through the use of a boronate affinity gel, we have developed an efficient route to the preparation of folate conjugated AMP with high yields and purity. Affi-Gel boronate is an affinity resin that selectively binds nucleoside and nucleoside derivatives at pH>7.5 and releases them at pH<6.5. This resin is used to efficiently bind and purify ribonucleotides such as AMP. This allows for the addition of a large excess of reactants and reagents in order to drive the reaction to completion and then allow easy purification without HPLC. The synthesis can be successfully scaled up to produce large quantities of AMP conjugates.

RNA detection using peptide-inserted Renilla luciferase.

A novel complementation system with short peptide-inserted-Renilla luciferase (PI-Rluc) and split-RNA probes was constructed for noninvasive RNA detection. The RNA binding peptides HIV-1 Rev and BIV Tat were used as inserted peptides. They display induced fit conformational changes upon binding to specific RNAs and trigger complementation or discomplementation of Rluc. Split-RNA probes were designed to reform the peptide binding site upon hybridization with arbitrarily selected target RNA. This set of recombinant protein and split-RNA probes enabled a high degree of sensitivity in RNA detection. In this study, we show that the Rluc system is comparable to Fluc, but that its detection limit for arbitrarily selected RNA (at least 100 pM) exceeds that of Fluc by approximately two orders of magnitude.

Confocal analysis of Hedgehog morphogenetic gradient coupled with fluorescent in situ hybridization of hedgehog target genes.

Hedgehog (Hh) family members are secreted proteins that can act at short and long range to direct cell fate decisions during developmental processes. In both Drosophila and vertebrates, the morphogenetic gradient of Hh must be tightly regulated for correct patterning. The posttranslational modification of Hh by a cholesterol adduct participates in such regulation. We have shown that cholesterol modification is necessary for the controlled long-range activity of Drosophila Hh, as observed for its vertebrate counterpart Sonic Hh. The presence of cholesterol on Hh allows the observation of large apical punctuate structures of Hh (Hh-LPSs) at a distance from the Hh source both in embryos and in imaginal discs. The Hh-LPSs apical distribution reflects the Hh gradient and is temporally regulated. Hh gradient modulation is directly related to the dynamic expression of the Hh target gene serrate (ser), shown by immunofluorescent detection of Hh coupled with fluorescent in situ hybridization of ser.

Enzymatic labeling of nucleic acids.

Extensive knowledge of the enzymology involved in biosynthesis and degradation of nucleic acids has permitted the development of simple methods for labeling RNA and DNA with radioisotopes or biotin. These labeled probes are used primarily for hybridization to nucleic acid fragments of interest in a variety of applications. A complete description of the methods available for such labeling is beyond the scope of this manual, but contained within this unit are protocols for oligonucleotide-primed synthesis of radiolabeled and biotinylated DNA probes, in vitro synthesis of radiolabeled RNA, and end labeling of synthetic oligonucleotide probes.

Application of DNA topoisomerase-activated adapters to riboprobe synthesis.

Topoisomerase-activated adapters for rapid incorporation of the T7 promoter into PCR products were made by hybridizing synthetic oligonucleotides and activating vaccinia DNA topoisomerase I. The adapters were used to incorporate the T7 promoter sequence into PCR products amplified from cDNA and genomic DNA. Modified PCR products were used as templates to synthesize digoxigenin-labeled sense and cRNA probes by in vitro transcription with phage T7 RNA polymerase. The red/green cones were labeled by the antisense probe, but no specific signal was produced by the sense probe. These results demonstrate that topoisomerase-activated adapters provide a powerful and convenient tool for the rapid modification of PCR products.

Expression of alpha2A adrenoceptors during rat neocortical development.

Norepinephrine has been suggested to play a neurotrophic role during development and is present in the brain as early as embryonic day (E) 12. We have recently demonstrated that the alpha2A adrenoceptor subtype is widely expressed during times of neuronal migration and differentiation throughout the developing brain. Here, we report the temporal and spatial expression pattern of alpha2A adrenoceptors in neocortex during late embryonic and early postnatal development using in situ hybridization and receptor autoradiography. Functional alpha2 receptors in embryonic rat cortex were also detected using agonist stimulated [35S]GTPgammaS autoradiography. Both alpha2A mRNA and protein expression were strongly increased by E19 and E20, respectively. The increased expression was in the cortical plate and intermediate and subventricular zones, corresponding to tiers of migrating and differentiating neurons. This transient up-regulation of alpha2A adrenoceptors was restricted to the lateral neocortex. At E20, functional alpha2 adrenoceptors were also detected in deep layers of lateral neocortex. During the first week of postnatal development, the expression of alpha2A mRNA and protein changed markedly, giving rise to a more mature pattern of anatomical distribution. The temporal and spatial distribution of alpha2A adrenoceptors in developing neocortex is consistent with expression of functional proteins on migrating and differentiating layer IV to II neurons. These findings suggest that alpha2A receptors may mediate a neurotrophic effect of norepinephrine during fetal cortical development. The early delineation of the lateral neocortex, which will develop into somatosensory and auditory cortices, suggests an intrinsic regulation of alpha2A mRNA expression.

[Riboprobes: an alternative in the detection of viral sequences]. / Las ribosondas: una alternativa en la detección de secuencias virales.

The use of riboprobes for the detection of RNA viral sequences is analyzed. Subgenomic fragments of cDNA from poliovirus type 2, dengue virus type 4 and human immunodeficiency virus type 1, were inserted downstream SP6 and/or T7 promoters in the transcription vectors pGEM-4z or pSP64. RNAs obtained by in vitro transcription in the presence of UTP infinity (32P) were used as probes for the detection of RNA viral sequences from infected cell lines in slot and Northern blot assays. The poliovirus riboprobe (P2-221) was able to detect specific viral sequences; thus, it could be used for the detection of the virus in serum, as well as in residual waters. The human immunodeficiency virus riboprobe (HIV-378), detected viral sequences poly A+RNA from infected cells; thus it can be used as a confirmatory test or as a tool in basic research. Finally, the dengue virus riboprobes (D4-2819 and D4-1134) detected specifically dengue 4 virus; however the sensitivity of the detection could be significantly improved amplifying viral sequences by the polymerase chain reaction (PCR) prior to probe hybridization.

Rapid nonradioactive in situ hybridization for interleukin-2 mRNA with riboprobes generated using the polymerase chain reaction.

In situ hybridization is a technique with widespread application. However, its usefulness has been limited by the need for radioactive materials and the requirement for the DNA to be cloned onto an appropriate vector. We have utilized the polymerase chain reaction to directly incorporate a T7 RNA polymerase promoter sequence onto the cDNA for interleukin-2. Digoxigenin-labelled riboprobes were then synthesized using this PCR product as a template. The digoxigenin-labelled riboprobes were then used in non-radioactive in situ hybridization to detect messenger RNA for interleukin-2 in mitogen stimulated peripheral blood mononuclear cells. This methodology has the potential for widespread application in immunology and cytokine research.

Single-stranded RNA probes generated from PCR-derived DNA templates.

The following report outlines the use of the polymerase chain reaction (PCR) in combination with in vitro transcription to generate single-stranded radiolabelled RNA probes useful for nuclease protection and in situ hybridization experiments. Specific DNA fragments with bacteriophage promoter (T3 and/or T7) sequences at the 5' or 3' end are generated by repeated rounds of amplification. Following purification, these PCR-generated DNA products are used as templates for in vitro transcription with the correct DNA-dependent RNA polymerase. The resultant radiolabelled, single-stranded RNA (ssRNA) can be used for in situ hybridization, Southern or Northern blot analysis, and ribonuclease protection experiments. Sub-cloning or hydrolysis of large fragments is not required. Probes can be made from virtually any sequence using a variety of template sources.

Synthesis of RNA probes by the direct in vitro transcription of PCR-generated DNA templates.

We describe a novel method for the generation of RNA probes based on the direct in vitro transcription of DNA templates amplified by polymerase chain reaction (PCR) using primers with sequence hybrids between the target gene and those of the T7 and T3 RNA polymerases promoters. This method circumvents the need for cloning and allows rapid generation of strand-specific RNA molecules that can be used for the identification of genes in hybridization experiments. We have successfully applied this method to the identification of DNA sequences by Southern blot analysis and library screening.