RNA-based stable isotope probing (RNA-SIP) to unravel intestinal host-microbe interactions.

The RNA-SIP technology, introduced into molecular microbial ecology in 2002, is an elegant technique to link the structure and function of complex microbial communities, i.e. to identify microbial key-players involved in distinct degradation and assimilation processes under in-situ conditions. Due to its dependence of microbial RNA, this technique is particularly suited for environments with high numbers of very active, i.e. significantly RNA-expressing, bacteria. So far, it was mainly used in environmental studies using microbiotas from soil or water habitats. Here we outline and summarize our application of RNA-SIP for the identification of bacteria involved in the degradation and assimilation of prebiotic carbohydrates in intestinal samples of human and animal origin. Following an isotope label from a prebiotic substrate into the RNA of distinct bacterial taxa will help to better understand the functionality of these medically and economically important nutrients in an intestinal environment.

Multiplexed miRNA northern blots via hybridization chain reaction.

Northern blots enable detection of a target RNA of interest in a biological sample using standard benchtop equipment. miRNAs are the most challenging targets as they must be detected with a single short nucleic acid probe. With existing approaches, it is cumbersome to perform multiplexed blots in which several RNAs are detected simultaneously, impeding the study of interacting regulatory elements. Here, we address this shortcoming by demonstrating multiplexed northern blotting based on the mechanism of hybridization chain reaction (HCR). With this approach, nucleic acid probes complementary to RNA targets trigger chain reactions in which fluorophore-labeled DNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability of HCR allows multiple amplifiers to operate simultaneously and independently within a blot, enabling straightforward multiplexing. We demonstrate simultaneous detection of three endogenous miRNAs in total RNA extracted from 293T and HeLa cells. For a given target, HCR signal scales linearly with target abundance, enabling relative and absolute quantitation. Using non-radioactive HCR, sensitive and selective miRNA detection is achieved using 2'OMe-RNA probes. The HCR northern blot protocol takes ∼1.5 days independent of the number of target RNAs.

Novel Biochemical Tools for Probing HIV RNA Structure.

Functional analysis of viral RNA requires knowledge of secondary structure arrangements and tertiary base interactions. Thus, high-throughput and comprehensive methods for assessing RNA structure are highly desirable. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) has proven highly useful for modeling the secondary structures of HIV and other retroviral RNAs in recent years. This technology is not without its limitations however, as SHAPE data can be severely compromised when the RNA under study is structurally heterogeneous. In addition, the method reveals little information regarding the three-dimensional (3D) organization of an RNA. This chapter outlines four detailed SHAPE-related methodologies that circumvent these limitations. "Ensemble" and "in-gel" variations of SHAPE permit structural analysis of individual conformers within structurally heterogeneous mixtures of RNA, while probing strategies that utilize "through-space" cleavage reagents such as methidiumpropyl-EDTA (MPE) and peptides appended with an ATCUN (amino terminal copper/nickel binding motif) can provide insight into 3D organization. Combinational application of these techniques provides a formidable arsenal for exploring the structures of HIV RNAs and associated nucleoprotein complexes.

Application of bioinformatics in probe design enables detection of enteroviruses on different taxonomic levels by advanced in situ hybridization technology.

BACKGROUND: Enteroviral infections are common, affecting humans across all age groups. RT-PCR is widely used to detect these viruses in clinical samples. However, there is a need for sensitive and specific in situ detection methods for formalin-fixed tissues, allowing for the anatomical localization of the virus and identification of its serotype. OBJECTIVES: The aim was to design novel enterovirus probes, assess the impact of probe design for the detection and optimize the new single molecule in situ hybridization technology for the detection of enteroviruses in formalin-fixed paraffin-embedded samples. STUDY DESIGN: Four enterovirus RNA-targeted oligonucleotide RNA probes - two probes for wide range enterovirus detection and two for serotype-targeted detection of Coxsackievirus B1 (CVB1) - were designed and validated for the commercially available QuantiGene ViewRNA in situ hybridization method. The probe specificities were tested using a panel of cell lines infected with different enterovirus serotypes and CVB infected mouse pancreata. RESULTS: The two widely reactive probe sets recognized 19 and 20 of the 20 enterovirus serotypes tested, as well as 27 and 31 of the 31 CVB1 strains tested. The two CVB1 specific probe sets detected 30 and 14 of the 31 CVB1 strains, with only minor cross-reactivity to other serotypes. Similar results were observed in stained tissues from CVB -infected mice. CONCLUSIONS: These novel in-house designed probe sets enable the detection of enteroviruses from formalin-fixed tissue samples. Optimization of probe sequences makes it possible to tailor the assay for the detection of enteroviruses on the serotype or species level.

Alternations in genes expression of pathway signaling in esophageal tissue with atresia: results of expression microarray profiling.

Esophageal atresia (EA) is a congenital defect of the esophagus involving the interruption of the esophagus with or without connection to the trachea (tracheoesophageal fistula [TEF]). EA/TEF may occur as an isolated anomaly, may be part of a complex of congenital defects (syndromic), or may develop within the context of a known syndrome or association. The molecular mechanisms underlying the development of EA are poorly understood. It is supposed that a combination of multigenic factors and epigenetic modification of genes play a role in its etiology. The aim of our work was to assess the human gene expression microarray study in esophageal tissue samples. Total RNA was extracted from 26 lower pouches of esophageal tissue collected during thoracoscopic EA repair in neonates with the isolated (IEA) and the syndromic form (SEA). We identified 787 downregulated and 841 upregulated transcripts between SEA and controls, and about 817 downregulated and 765 upregulated probes between IEA and controls. Fifty percent of these genes showed differential expression specific for either IEA or SEA. Functional pathway analysis revealed substantial enrichment for Wnt and Sonic hedgehog, as well as cytokine and chemokine signaling pathways. Moreover, we performed reverse transcription polymerase chain reaction study in a group of SHH and Wnt pathways genes with differential expression in microarray profiling to confirm the microarray expression results. We verified the altered expression in SFRP2 gene from the Wnt pathway as well as SHH, GLI1, GLI2, and GLI3 from the Sonic hedgehog pathway. The results suggest an important role of these pathways and genes for EA/TEF etiology.

MicroRNA in situ hybridization on whole-mount preimplantation embryos.

Whole-mount in situ hybridization (WISH) using antisense probes is widely used to visualize RNA sequences in embryos and to determine the precise site of expression in the different cells or tissues. The target sequence is hybridized with an antisense RNA probe, followed by visual or fluorescence detection to measure the site and level of expression. However, the detection of short RNA molecules is hampered by the reduced stringency of the probes for short transcripts. Here, we describe a procedure for WISH detection of short RNA molecules, like miRNAs, in mammalian preimplantation embryos using LNA-modified probes with high sensitivity and specificity.

Whole-mount in situ hybridization using DIG-labeled probes in planarian.

In recent years freshwater flatworms (planarian) have become a powerful model for studies of regeneration and stem cell biology. Whole-mount in situ hybridization (WISH) and fluorescent in situ hybridization (FISH) are key and most commonly used techniques to determine and visualize gene expression patterns in planaria. Here, we present the established version of whole-mount in situ hybridization (WISH) and whole-mount fluorescence in situ hybridization (WFISH) protocol optimized over the last years by several labs from the rapidly growing planaria field and give an overview of recently introduced modifications which can be critical in the study of low abundant transcripts.

In situ hybridization on whole-mount zebrafish embryos and young larvae.

The in situ hybridization uses a labeled complementary RNA strand to localize a specific mRNA sequence in a tissue. This method is widely used to describe the spatial and temporal expression patterns of developmentally regulated genes. Here we describe a technique that employs in vitro synthesized RNA tagged with digoxigenin uridine-5'-triphosphate (UTP) to determine expression of genes on whole-mount zebrafish embryos and young larvae. Following hybridization, the localization of the specific transcript is visualized immunohistochemically using an anti-digoxigenin antibody conjugated to alkaline phosphatase that hydrolyzes the 5-bromo-4-chloro-3-indolyl phosphate (BCIP) to 5-bromo-4-chloro-3-indole and inorganic phosphate. 5-Bromo-4-chloro-3-indole can be oxidized by nitro blue tetrazolium (NBT), which forms an insoluble dark blue diformazan precipitate after reduction.This protocol has been used for performing large-scale analyses of the spatial and temporal expression of the zebrafish genome, resulting in the description of more than 8,400 expression patterns that are available at the zebrafish information network (ZFIN.org) in the gene expression section.

Sensitive and specific in situ hybridization for early drug discovery.

High-throughput analyses of gene expression such as microarrays and RNA-sequencing are widely used in early drug discovery to identify disease-associated genes. To further characterize the expression of selected genes, in situ hybridization (ISH) using RNA probes (riboprobes) is a powerful tool to localize mRNA expression at the cellular level in normal and diseased tissues, especially for novel drug targets, where research tools like specific antibodies are often lacking.We describe a sensitive ISH protocol using radiolabelled riboprobes suitable for both paraffin-embedded and cryo-preserved tissue. The riboprobes are generated by in vitro transcription using PCR products as templates, which is less time consuming compared to traditional transcription from linearized plasmids, and offers a relatively simple way to generate several probes per gene, e.g., for splice variant analyses. To ensure reliable ISH results, we have incorporated a number of specificity controls in our standard experimental setup. We design antisense probes to cover two non-overlapping parts of the gene of interest, and use the corresponding sense probes as controls for unspecific binding. Probes are furthermore tested on sections of paraffin-embedded or cryo-preserved positive and negative control cells with known gene expression. Our protocol thus provides a method for sensitive and specific ISH, which is suitable for target validation and characterization in early drug discovery.

Multiplexed miRNA fluorescence in situ hybridization for formalin-fixed paraffin-embedded tissues.

Multiplexed miRNA fluorescence in situ hybridization (miRNA FISH) is an advanced method for visualizing differentially expressed miRNAs, together with other reference RNAs, in archival tissues. Some miRNAs are excellent disease biomarkers due to their abundance and cell-type specificity. However, these short RNA molecules are difficult to visualize due to loss by diffusion, probe mishybridization, and signal detection and signal amplification issues. Here, we describe a reliable and adjustable method for visualizing and normalizing miRNA signals in formalin-fixed paraffin-embedded (FFPE) tissue sections.

Explanatory chapter: nuclease protection assays.

Nuclease protection assays are a highly sensitive, solution-based technique used to detect and quantify specific RNA targets from complex RNA mixtures. Today, this technique is frequently performed using kits, and the following chapter will explain the principles of how these kits work and some considerations to keep in mind when using them.

High resolution fluorescent in situ hybridization in Drosophila.

Tissue-specific gene expression is a major determinant in the elaboration of cells with distinctive phenotypes and functions, which is crucial for the development and homeostasis of multicellular organisms. Fluorescent in situ hybridization (FISH) is a powerful method for assessing the expression and localization properties of RNA at subcellular resolution in whole mount organism and tissue specimens. This chapter describes a high-resolution FISH protocol for the detection of RNA expression and localization dynamics in embryos and tissues of the fruit fly, Drosophila melanogaster. The approach utilizes tyramide signal amplification (TSA) for enhanced sensitivity and resolution in the detection of coding and noncoding RNAs, for the codetection of different RNA species or of RNA and a protein marker of interest. Furthermore, the protocol outlines details for conducting FISH in microtiter plates, which greatly enhances the throughput, practicality, and economy of the procedure.

Analysis of messenger RNA expression by in situ hybridization using RNA probes synthesized via in vitro transcription.

The analysis of the spatial patterning of mRNA expression is critically important for assigning functional and physiological significance to a given gene product. Given the tens of thousands of mRNAs in the mammalian genome, a full assessment of individual gene functions would ideally be overlaid upon knowledge of the specific cell types expressing each mRNA. In situ hybridization approaches represent a molecular biological/histological method that can reveal cellular patterns of mRNA expression. Here, we present detailed procedures for the detection of specific mRNAs using radioactive RNA probes in tissue sections followed by autoradiographic detection. These methods allow for the specific and sensitive detection of spatial patterns of mRNA expression, thereby linking mRNA expression with cell type and function. Radioactive detection methods also facilitate semi-quantitative analyses of changes in mRNA gene expression.

Using RNA FISH to study gene expression during mammalian meiosis.

During mouse meiosis, gene expression and homologous synapsis are intimately linked. Chromosomes that fail to synapse at the zygotene-pachytene transition become transcriptionally silenced by a process called Meiotic Silencing of Unsynapsed Chromatin (MSUC), and this silencing (or defects in it) may in turn cause germ cell losses and infertility. Gene transcription at the chromosomal level can be readily observed using RNA fluorescence in-situ hybridisation (FISH), and this approach allows one to directly compare expression at a specific locus with the synaptic status of the chromosome domain on which it resides. Here we describe a protocol for carrying out RNA FISH on male meiotic cells, together with detail on the important controls and common problems associated with this technique.

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.

Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization.

Here we describe a fluorescence in situ hybridization protocol that allows for the detection of two mRNA species in fresh frozen brain tissue sections. This protocol entails the simultaneous and specific hybridization of hapten-labeled riboprobes to complementary mRNAs of interest, followed by probe detection via immunohistochemical procedures and peroxidase-mediated precipitation of tyramide-linked fluorophores. In this protocol we describe riboprobes labeled with digoxigenin and biotin, though the steps can be adapted to labeling with other haptens. We have used this approach to establish the neurochemical identity of sensory-driven neurons and the co-induction of experience-regulated genes in the songbird brain. However, this procedure can be used to detect virtually any combination of two mRNA populations at single-cell resolution in the brain, and possibly other tissues. Required controls, representative results and troubleshooting of important steps of this procedure are presented. After tissue sections are obtained, the total length of the procedure is 2-3 d.

MEC-2 and MEC-6 in the Caenorhabditis elegans sensory mechanotransduction complex: auxiliary subunits that enable channel activity.

The ion channel formed by the homologous proteins MEC-4 and MEC-10 forms the core of a sensory mechanotransduction channel in Caenorhabditis elegans. Although the products of other mec genes are key players in the biophysics of transduction, the mechanism by which they contribute to the properties of the channel is unknown. Here, we investigate the role of two auxiliary channel subunits, MEC-2 (stomatin-like) and MEC-6 (paraoxonase-like), by coexpressing them with constitutively active MEC-4/MEC-10 heteromeric channels in Xenopus oocytes. This work extends prior work demonstrating that MEC-2 and MEC-6 synergistically increase macroscopic current. We use single-channel recordings and biochemistry to show that these auxiliary subunits alter function by increasing the number of channels in an active state rather than by dramatically affecting either single-channel properties or surface expression. We also use two-electrode voltage clamp and outside-out macropatch recording to examine the effects of divalent cations and proteases, known regulators of channel family members. Finally, we examine the role of cholesterol binding in the mechanism of MEC-2 action by measuring whole-cell and single-channel currents in MEC-2 mutants deficient in cholesterol binding. We suggest that MEC-2 and MEC-6 play essential roles in modulating both the local membrane environment of MEC-4/MEC-10 channels and the availability of such channels to be gated by force in vivo.

Whole-mount identification of gene transcripts in aphids: protocols and evaluation of probe accessibility.

In situ hybridization has become a powerful tool for detecting the temporal and spatial distribution of gene transcripts in prokaryotes and eukaryotes. We report an efficient protocol for whole-mount identification of the expression of mRNAs in the parthenogenetic pea aphid Acyrthosiphon pisum, an emerging model organism with a growing accumulation of genome sequencing data. In addition to steps common for most animal in situ hybridization protocols, we describe processing methods specific to aphids, the accessibility of antisense riboprobes of different lengths in whole-mounted aphids, and signal intensity versus probe lengths. To find substrate combinations that clearly contrast single and double in situ signals in A. pisum, we tested our protocols using riboprobes constructed from two conserved germline markers, Apvasa and Apnanos, and examined colocalized signals in the germaria and developing oocytes. Finally, we propose conditions for stringent permeabilization that may be applied to tissues deep within the aphid embryo.

Algorithmic approaches to selecting control clones in DNA array hybridization experiments.

We study the problem of selecting control clones in DNA array hybridization experiments. The problem arises in the OFRG method for analyzing microbial communities. The OFRG method performs classification of rRNA gene clones using binary fingerprints created from a series of hybridization experiments, where each experiment consists of hybridizing a collection of arrayed clones with a single oligonucleotide probe. This experiment produces analog signals, one for each clone, which then need to be classified, that is, converted into binary values 1 and 0 that represent hybridization and non-hybridization events. In addition to the sample rRNA gene clones, the array contains a number of control clones needed to calibrate the classification procedure of the hybridization signals. These control clones must be selected with care to optimize the classification process. We formulate this as a combinatorial optimization problem called Balanced Covering. We prove that the problem is NP-hard, and we show some results on hardness of approximation. We propose approximation algorithms based on randomized rounding, and we show that, with high probability, our algorithms approximate well the optimum solution. The experimental results confirm that the algorithms find high quality control clones. The algorithms have been implemented and are publicly available as part of the software package called CloneTools.

Selection of aptamers for molecular recognition and characterization of cancer cells.

In this paper, we describe a new way to generate molecular probes for specific recognition of cancer cells. Molecular medicine will require a large number of probes for molecular recognition and characterization of a variety of diseased cells. Aptamers, single-stranded DNA/RNA probes, are poised to become a chemist's antibody and have the potential to serve as molecular probes for a variety of biomedical applications. By applying newly developed cell-SELEX (cell-based systematic evolution of ligands by exponential enrichment) against whole living cells, panels of aptamers have been evolved from an initial DNA library to characterize target cells at the molecular level. Ramos cells, a B-cell lymphoma cell line, were used as target cells for the generation of effective molecular probes. By taking advantages of the repetitive and broad enrichment strategy, the selected aptamers could bind to target cells and other closely related cell lines in variant patterns with an equilibrium dissociation constant (Kd) in the nanomolar range. Some aptamers could also specifically recognize the target lymphoma cells mixed with normal human bone marrow aspirates. The cell-based SELEX is simple, fast, and robust. The strategies used here will be highly useful for aptamer selection against complex target samples in order to generate a large number of aptamers in a variety of biomedical and biotechnological applications, paving the way for molecular diagnosis, therapy, and biomarker discovery.