Whole Mount In Situ Hybridization in Murine Tissues.

Whole mount in situ hybridization is a sensitive method used to characterize the spatial and temporal expression of RNA transcripts throughout an entire tissue. This method is an excellent tool for studying gene expression during embryonic development. Here, we describe a procedure for digoxigenin labeled in situ hybridization on whole embryos.

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.

Purification of cardiomyocytes from differentiating pluripotent stem cells using molecular beacons that target cardiomyocyte-specific mRNA.

BACKGROUND: Although methods for generating cardiomyocytes from pluripotent stem cells have been reported, current methods produce heterogeneous mixtures of cardiomyocytes and noncardiomyocyte cells. Here, we report an entirely novel system in which pluripotent stem cell-derived cardiomyocytes are purified by cardiomyocyte-specific molecular beacons (MBs). MBs are nanoscale probes that emit a fluorescence signal when hybridized to target mRNAs. METHOD AND RESULTS: Five MBs targeting mRNAs of either cardiac troponin T or myosin heavy chain 6/7 were generated. Among 5 MBs, an MB that targeted myosin heavy chain 6/7 mRNA (MHC1-MB) identified up to 99% of HL-1 cardiomyocytes, a mouse cardiomyocyte cell line, but <3% of 4 noncardiomyocyte cell types in flow cytometry analysis, which indicates that MHC1-MB is specific for identifying cardiomyocytes. We delivered MHC1-MB into cardiomyogenically differentiated pluripotent stem cells through nucleofection. The detection rate of cardiomyocytes was similar to the percentages of cardiac troponin T- or cardiac troponin I-positive cardiomyocytes, which supports the specificity of MBs. Finally, MHC1-MB-positive cells were sorted by fluorescence-activated cell sorter from mouse and human pluripotent stem cell differentiating cultures, and ≈97% cells expressed cardiac troponin T or cardiac troponin I as determined by flow cytometry. These MB-based sorted cells maintained their cardiomyocyte characteristics, which was verified by spontaneous beating, electrophysiological studies, and expression of cardiac proteins. When transplanted in a myocardial infarction model, MB-based purified cardiomyocytes improved cardiac function and demonstrated significant engraftment for 4 weeks without forming tumors. CONCLUSIONS: We developed a novel cardiomyocyte selection system that allows production of highly purified cardiomyocytes. These purified cardiomyocytes and this system can be valuable for cell therapy and drug discovery.

In situ hybridization detection of microRNAs.

MicroRNAs (miRNAs) are endogenous approximately 22 nucleotide RNAs that play critical roles in many cellular processes including cell differentiation, proliferation, and apoptosis. The analysis of spatiotemporal expression of miRNAs is important for dissecting their roles in development and during physiological/pathophysiological processes. In situ hybridization is a powerful technology that allows cellular localization. However, the detection of miRNAs by in situ hybridization has been challenging because of the low affinity of conventional RNA or DNA probes due to the small sizes of miRNAs. Here, we describe a protocol for miRNA in situ hybridization on mouse tissue cryosections using locked nucleic acid (LNA) probes. LNA probes demonstrate a much higher affinity to their complimentary RNAs compared to conventional RNA and DNA oligo probes, which allow detection of miRNAs in tissue sections with excellent specificity.

Isolation of mRNA from specific tissues of Drosophila by mRNA tagging.

To study the function of specific cells or tissues using genomic tools like microarray analyses, it is highly desirable to obtain mRNA from a homogeneous source. However, this is particularly challenging for small organisms, like Caenorhabditis elegans and Drosophila melanogaster. We have optimized and applied a new technique, mRNA tagging, to isolate mRNA from specific tissues of D.melanogaster. A FLAG-tagged poly(A)-binding protein (PABP) is expressed in a specific tissue and mRNA from that tissue is thus tagged by the recombinant PABP and separated from mRNA in other tissues by co-immunoprecipitation with a FLAG-tag specific antibody. The fractionated mRNA is then amplified and used as probe in microarray experiments. As a test system, we employed the procedures to identify genes expressed in Drosophila photoreceptor cells. We found that most known photoreceptor cell-specific mRNAs were identified by mRNA tagging. Furthermore, at least 11 novel genes have been identified as enriched in photoreceptor cells. mRNA tagging is a powerful general method for profiling gene expression in specific tissues and for identifying tissue-specific genes.

RNA analysis by nuclease protection.

Nuclease protection assays (S1 nuclease protection and RNase protection) are extremely sensitive procedures for detection and quantitation of mRNA species in complex mixtures of total cellular RNA. These assays are well suited for mapping positions of external and internal junctions in RNA, such as transcription initiation and termination sites and intron/exon boundaries, and to discriminate between closely related targets by using probes designed to span the regions where the related genes differ the most. Also, because the size of the probes used in nuclease protection assays is a variable chosen by the investigator, probes may be designed to protect fragments of different sizes. This feature permits the simultaneous analysis of several different mRNAs in the same total RNA sample. In this unit, a method is included for RNase protection of target mRNA sequences, including hybridization of the probe to the target sequence, details of the actual protection assay, and detection of reaction products. An alternative method is provided for performing the RNase protection assay on a microvolume scale, which is useful when there are many samples to be analyzed. Support protocols describe synthesis and gel purification of labeled RNA probes; preparation of RNase-free yeast RNA, which acts as an aid in the quantitative precipitation of newly synthesized probe; and quantitation of target mRNA. A method describing S1 nuclease protection of target mRNA using either RNA or DNA probes is also included. Additional support protocols provide instructions for the preparation of radiolabeled DNA probes by primer-extension of double-stranded plasmid or PCR product using Klenow fragment of E. coli DNA polymerase I or Taq or Tth polymerase in a thermal cycler. Another radiolabeling method details 5' end labeling of oligodeoxynucleotides and oligoribonucleotides using T4 polynucleotide kinase. Additionally, a method is described for mapping transcription start sites using the S1 nuclease protection assay.

Detection of mutations by RNase cleavage.

Ribonucleases can specifically recognize and cleave RNA at the site of sequence mismatches in RNA-DNA or RNA-RNA hybrids. The cleavage products are then characterized by gel electrophoresis. In this unit, a procedure is presented for RNase cleavage of (32)P-labeled riboprobes (transcribed from a cloned copy of the normal sequence) that have been annealed to amplified sequences of a candidate gene or cDNA obtained from affected individuals. A Support Protocol explains how to prepare riboprobes from a genomic or cDNA template obtained from a nonmutant individual. An alternate protocol describes cleavage of RNARNA hybrids using a nonisotopic RNase cleavage mutation assay. Sequential PCR and in vitro transcription steps generate sufficient quantities of duplex RNA targets so that the cleavage products can be detected on a gel by ethidium bromide staining. The unit also discusses the use of alternative ribonucleases for cleaving singlebase mismatches.

Detailed characterization of the posttranscriptional gene-silencing-related small RNA in a GUS gene-silenced tobacco.

Posttranscriptional gene-silencing phenomena such as cosuppression and RNA interference are associated with the occurrence of small, about 21-23 nt short RNA species homologous to the silenced gene. We here show that the small RNA present in silenced transgenic plants can easily be detected in total RNA isolated according to standard procedures. This will allow for the development of routine and early screenings for the presence of small RNA species and, therefore, gene silencing in transgenic plants. We further demonstrate that the small RNA fraction can be visualized with the SYBR Green II RNA stain, isolated from a gel, labeled and used as a specific probe. Using these approaches, we have fine-mapped the sequences of the GUS gene that are represented in the small RNA fraction of a GUS-silenced tobacco line containing an inverted repeat of the GUS gene. In this tobacco line, the silencing-associated small RNA is a mixture of fragments that cover the 3' two-thirds of the GUS coding region. The 5' coding and the 3' noncoding ends of the GUS mRNA are not represented in the small RNA fraction. The RNA fragments are not likely to be a primary synthesis product of an RNA-dependent RNA polymerase, but rather degradation products from nuclease activity. Surprisingly, RNA isolated from wild-type, untransformed plants showed the presence of a similar-sized small RNA pool. This might indicate the existence of small RNA species from putative endogenous genes that are down regulated by a similar posttranscriptional gene-silencing mechanism. The possibility of isolating and labeling the small RNA pool from wild-type plants will provide a way to identify and study such putative genes.

Expression of ethylene biosynthetic genes in Actinidia chinensis fruit.

The fruit of Actinidia chinensis, a diploid relative of kiwifruit, showed an increased rate of ripening in response to the application of exogenous ethylene. Moreover, late in ripening the fruit produced a burst of ethylene biosynthesis. Thus ripening is climacteric, and there is a clear temporal separation of ethylene sensitivity and ethylene production. RNase protection assays were used to monitor transcript levels of ethylene biosynthetic genes during fruit development and ethylene-induced ripening. The application of exogenous ethylene correlated with increased transcript levels for three different S-adenosyl-L-methionine (SAM) synthetase genes and for the 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene family. Transcription of an ACC synthase gene was not affected by exogenous ethylene. However, ACC synthase transcript levels increased during subsequent ethylene production by the fruit, consistent with this being the control step for the onset of climacteric ethylene production. ACC oxidase transcripts increased significantly both prior to and during climacteric ethylene production, while only one of the three SAM synthetase transcripts was induced during the late ethylene burst. We propose that the regulation of SAM synthetase transcripts by ethylene may occur as part of the methionine salvage pathway.

Simplified riboprobe purification using translucent straws as gel tubes.

Gel purification of radioactive riboprobes enhances the quality of the ribonuclease protection assay. A simple and effective method for riboprobe purification is described. The method uses acrylamide gels in plastic tubes to achieve electrophoretic separation of the RNA polymerase products.

Applications of laser scanning microscopy for analysis of aquatic microhabitats.

Laser scanning microscopy was used 1) to analyze Mn oxide-encrusted biofilms and particles in marine Mn-oxidizing enrichment cultures, 2) to optimize fluorescence in situ hybridization protocols of 16S ribosomal RNA-targeted oligonucleotide signature probes for single bacterial cell identification in particles from a wetland, and 3) to develop a combined immunofluorescence-microautoradiography procedure for analysis of the distribution of 14C-labeled organic compounds and 14C-mineralizing bacteria in groundwater seep sediments. The results demonstrated the wide applicability and benefits of using laser scanning microscopy for analysis of complex microbial assemblages.

Quantification of biotinylated RNA probes for in situ hybridization using chemiluminescence.

For reliable detection of mRNA by non-radioactive in situ hybridization, calibration and standardization of the individual steps involved are essential. We describe a method that allows determination of the size and integrity as well as quantification of biotinylated RNA probes in a single experiment. Serial dilutions of biotinylated RNA probes generated by promoter-mediated in vitro transcription were size-separated by gel electrophoresis in the presence of known amounts of 5'-biotinylated oligomers which served as internal standard. Following immobilization onto nylon membranes and visualization by chemiluminescence, optical densities of probes and internal standards were measured by densitometry and analysed by linear regression. RNA probes complementary to the human homeobox genes HOX-C6, -C8 and -C9 were quantified. Four different 5'-biotinylated oligomers (20, 35, 50 and 75 bases) were tested as internal standards. Concerning the separation of probe and oligomer in the gel, transfer properties and efficiency of binding to the membrane, the oligomer of 35 bases was found to be the best internal standard with highest reproducibility. Comparison of probe concentration by spectrophotometry and the described method showed a good correlation, indicating that our method is a reliable assay for quantitative and qualitative control of biotin-labelled probes.

Preparation of a specific RNA probe for detection of Mycobacterium paratuberculosis and diagnosis of Johne's disease.

A species-specific recombinant clone (F57) was obtained from a genomic library of Mycobacterium paratuberculosis in the transcription vector pGem 3Z. This clone proved to be specific for all mycobacteria tested, including M. avium, and was able to recognize all of the tested M. paratuberculosis strains isolated from animals and humans (patients with Crohn's disease). The F57 insert was sequenced and a segment of 620 bp with a G + C content of 58.9% was identified. Comparison of the sequence with sequences in the EMBL and UGEN data banks revealed the uniqueness of the F57 sequence, which had no resemblance to other known genes.

[Methodological approaches to detecting mRNA in histological sections]. / Metodicheskie podkhody k vyiavleniiu mRNK na gistologicheskikh srezakh.

This paper reviews methodological approaches to detection of mRNA on histological sections using in situ hybridization. Alongside with the review of literature we present a detailed description of our own modification of this technique, based on the use of paraffin sections of the material fixed by replacement in the frozen state. Particular attention is given to methodological problems facing the researcher who is using in situ hybridization for the first time.

Characterization of a nucleic acid probe for the diagnosis of human coronavirus 229E infections.

A cDNA copy of the HCV229E nucleocapsid protein gene was isolated and characterized. Sequence analysis predicts a nucleocapsid polypeptide of 389 amino acids with a molecular weight (mol. wt.) of 43,450. Single strand RNA probes derived from the cDNA copy hybridize specifically to HCV229E RNA and approximately 50 pg of intracellular viral RNA can be readily detected. The application of nucleic acid hybridization as a routine procedure for the diagnosis of HCV229E infection is discussed.

Transcriptional and posttranscriptional regulation of sexually differentiated rat liver cytochrome P-450 by growth hormone.

The female specific cytochrome P-45015 beta and the male specific cytochrome P-45016 alpha are the major constitutive P-450 forms in rat liver. Their expression is pretranslationally regulated by the sex specific plasma pattern of GH. To measure the mRNA levels of these hemoproteins in studies on their regulation and expression we set up specific solution hybridization assays with in vitro transcribed cRNA probes. An extensive tissue distribution study showed that both P-45015 beta and P-45016 alpha are specifically expressed in the liver. Investigation of the developmental regulation of respective mRNA showed that the onset of sexual differentiation of P-45015 beta and P-45016 alpha expression coincides with the start of puberty. To assess the level(s) of the pretranslational GH regulation of P-45015 beta and P-45016 alpha nuclear run-on transcription analysis was performed. Sex characteristic administration of GH to hypophysectomized rats indicated that GH exerts its dual effects via both transcriptional and postranscriptional mechanisms.

Somatic mutation and transcriptional deregulation of myc in endemic Burkitt's lymphoma disease: heptamer-nonamer recognition mistakes?

We examined the structure and expression of the myc protooncogene in DNA extracted from a primary (uncultured) endemic Burkitt's lymphoma sample designated eBL3. Dot and Northern (RNA) blot analyses demonstrated extreme levels of myc RNA in the eBL3 sample. Nearly complete sequence data of the altered myc locus isolated from eBL3 DNA demonstrated extensive mutations (duplications, insertions, and deletions) in critical myc regulatory regions. Taken together, the data support the idea that myc transcriptional deregulation in Burkitt's lymphoma disease may be a consequence of the position and number of mutations produced within and around the myc locus. Furthermore, the myc exon-1-intron-1 hypermutable PvuII site is part of a potential heptamer-nonamer recognition sequence, suggesting a mechanism for mutation in endemic Burkitt's lymphoma disease.