Analysis of microRNA expression by in situ hybridization with RNA oligonucleotide probes

In situ hybridization is an important tool for analyzing gene expression and developing hypotheses about gene functions. The discovery of hundreds of microRNA (miRNA) genes in animals has provided new challenges for analyzing gene expression and functions. The small size of the mature miRNAs ( approximately 20-24 nucleotides in length) presents difficulties for conventional in situ hybridization methods. However, we have described a modified in situ hybridization method for detection of mammalian miRNAs in tissue sections, based upon the use of RNA oligonucleotide probes in combination with highly specific wash conditions. Here, we present detailed procedures for detection of miRNAs in tissue sections or cultured cells. The methods described can utilize either nonradioactive hapten-conjugated probes that are detected by enzyme-coupled antibodies, or radioactively labeled probes that are detected by autoradiography. The ability to visualize miRNA expression patterns in tissue sections provides an additional tool for the analyses of miRNA expression and function. In addition, the use of radioactively labeled probes should facilitate quantitative analyses of changes in miRNA gene expression.     

In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.     

Loading and transfer control for Northern hybridization.

We report a simple and inexpensive method to quantitate loading and transfer of RNA and to examine RNA integrity for use with Northern hybridization. This technique involves quantitation by computer-assisted video densitometry of a negative photograph of 28S and 18S rRNA from ethidium bromide-stained RNA fixed to nylon membranes. This method eliminates the issue of variability of expression of "housekeeping" genes and the need for a second round of hybridization to quantitate control probes.     

Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes

We describe here a new method for highly efficient detection of microRNAs by northern blot analysis using LNA (locked nucleic acid)-modified oligonucleotides. In order to exploit the improved hybridization properties of LNA with their target RNA molecules, we designed several LNA-modified oligonucleotide probes for detection of different microRNAs in animals and plants. By modifying DNA oligonucleotides with LNAs using a design, in which every third nucleotide position was substituted by LNA, we could use the probes in northern blot analysis employing standard end-labelling techniques and hybridization conditions. The sensitivity in detecting mature microRNAs by northern blots was increased by at least 10-fold compared to DNA probes, while simultaneously being highly specific, as demonstrated by the use of different single and double mismatched LNA probes. Besides being highly efficient as northern probes, the same LNA-modified oligonucleotide probes would also be useful for miRNA in situ hybridization and miRNA expression profiling by LNA oligonucleotide microarrays.     

The effect of nucleobase-specific fluorescence quenching on in situ hybridization with rRNA-targeted oligonucleotide probes

Oligonucleotide probes labeled with fluorescent dyes are used in a variety of in situ applications to detect specific DNA or RNA molecules. It has been described that probe fluorescence might be quenched upon hybridization in a sequence specific way. Here, a set of 17 oligonuleotides labeled with 6-carboxyfluorescein was used to examine the relevance of nucleotide specific quenching for fluorescence in situ hybridization (FISH) to whole fixed bacterial cells. Probes quenched upon hybridization to a guanine-rich region of purified RNA in solution were not quenched upon FISH. Among other factors the high protein concentration within cells may prevent quenching of probe fluorescence in situ.     

On-chip hybridization kinetics for optimization of gene expression experiments

DNA microarray technology is a powerful tool for getting an overview of gene expression in biological samples. Although the successful use of microarray-based expression analysis was demonstrated in a number of applications, the main problem with this approach is the fact that expression levels deduced from hybridization experiments do not necessarily correlate with RNA concentrations. Moreover oligonucleotide probes corresponding to the same gene can give different hybridization signals. Apart from cross-hybridizations and differential splicing, this could be due to secondary structures of probes or targets. In addition, for low-copy genes, hybridization equilibrium may be reached after hybridization times much longer than the one commonly used (overnight, i.e., 15 h). Thus, hybridization signals could depend on kinetic properties of the probe, which may vary between different oligonucleotide probes immobilized on the same microarray. To validate this hypothesis, on-chip hybridization kinetics and duplex thermostability analysis were performed using oligonucleotide microarrays containing 50-mer probes corresponding to 10 mouse genes. We demonstrate that differences in hybridization kinetics between the probes exist and can influence the interpretation of expression data. In addition, we show that using on-chip hybridization kinetics, quantification of targets is feasible using calibration curves.     

Label-free detection of 16S ribosomal RNA hybridization on reusable DNA arrays using surface plasmon resonance imaging

In this paper, we describe the detection of bacterial cell-extracted 16S ribosomal RNA (rRNA) using an emerging technology, surface plasmon resonance (SPR) imaging of DNA arrays. Surface plasmon resonance enables detection of molecular interactions on surfaces in response to changes in the index of refraction, therefore eliminating the need for a fluorescent or radioactive label. A variation of the more common SPR techniques, SPR imaging enables detection from multiple probes in a reusable array format. The arrays developed here contain DNA probes (15-21 bases) designed to be complementary to 16S rRNA gene sequences of Escherichia coli and Bacillus subtilis as well as to a highly conserved sequence found in rRNAs from most members of the domain Bacteria. We report species-specific hybridization of cell-extracted total RNA and in vitro transcribed 16S rRNA to oligonucleotide probes on SPR arrays. We tested multiple probe sequences for each species, and found that success or failure of hybridization was dependent upon probe position in the 16S rRNA molecule. It was also determined that one of the probes intended to bind 16S rRNA also bound an unknown protein. The amount of binding to these probes was quantified with SPR imaging. A detection limit of 2 micro g ml-1 was determined for fragmented E. coli total cellular RNA under the experimental conditions used. These results indicate the feasibility of using SPR imaging for 16S rRNA identification and encourage further development of this method for direct detection of other RNA molecules.     

Linear 2' O-Methyl RNA probes for the visualization of RNA in living cells

U1snRNA, U3snRNA, 28 S ribosomal RNA, poly(A) RNA and a specific messenger RNA were visualized in living cells with microinjected fluorochrome-labeled 2' O-Methyl oligoribonucleotides (2' OMe RNA). Antisense 2' OMe RNA probes showed fast hybridization kinetics, whereas conventional oligodeoxyribonucleotide (DNA) probes did not. The nuclear distributions of the signals in living cells were similar to those found in fixed cells, indicating specific hybridization. Cytoplasmic ribosomal RNA, poly(A) RNA and mRNA could hardly be visualized, mainly due to a rapid entrapment of the injected probes in the nucleus. The performance of linear probes was compared with that of molecular beacons, which due to their structure should theoretically fluoresce only upon hybridization. No improvements were achieved however with the molecular beacons used in this study, suggesting opening of the beacons by mechanisms other than hybridization. The results show that linear 2' OMe RNA probes are well suited for RNA detection in living cells, and that these probes can be applied for dynamic studies of highly abundant nuclear RNA. Furthermore, it proved feasible to combine RNA detection with that of green fluorescent protein-labeled proteins in living cells. This was applied to show co-localization of RNA with proteins and should enable RNA-protein interaction studies.     

Group-specific 16S rRNA hybridization probes for determinative and community structure studies of Butyrivibrio fibrisolvens in the rumen.

Oligonucleotide probes covering three phylogenetically defined groups of Butyrivibrio spp. were successfully designed and tested. The specificity of each probe was examined by hybridization to rRNAs from an assortment of B. fibrisolvens isolates as well as additional ruminal and nonruminal bacteria. The sensitivity of the hybridization method was determined by using one of the probes (probe 156). When RNA was extracted from a culture of OB156, the probe was able to detect target cells at densities as low as 10(4) cells/ml. When 10(4) or more target cells/ml were added to cattle rumen samples, detectable hybridization signals were obtained with 1,000 ng of total RNA loaded onto the nylon membrane. In contrast, the sensitivity was reduced to 10(6) target cells/ml at 100 ng of RNA per slot. The probes were used to type 19 novel Butyrivibrio isolates. The phylogenetic placement was confirmed by partial 16S rRNA gene sequencing. The use of the probes in community-based studies indicated that the Butyrivibrio groups examined in this paper did not represent a significant portion of the bacterial 16S rRNA pool in the rumen of the cattle, sheep, and deer examined.     

Linear 2′ O-Methyl RNA probes for the visualization of RNA in living cells

U1snRNA, U3snRNA, 28 S ribosomal RNA, poly(A) RNA and a specific messenger RNA were visualized in living cells with microinjected fluorochrome-labeled 2′ O-Methyl oligoribonucleotides (2′ OMe RNA). Antisense 2′ OMe RNA probes showed fast hybridization kinetics, whereas conventional oligodeoxyribonucleotide (DNA) probes did not. The nuclear distributions of the signals in living cells were similar to those found in fixed cells, indicating specific hybridization. Cytoplasmic ribosomal RNA, poly(A) RNA and mRNA could hardly be visualized, mainly due to a rapid entrapment of the injected probes in the nucleus. The performance of linear probes was compared with that of molecular beacons, which due to their structure should theoretically fluoresce only upon hybridization. No improvements were achieved however with the molecular beacons used in this study, suggesting opening of the beacons by mechanisms other than hybridization. The results show that linear 2′ OMe RNA probes are well suited for RNA detection in living cells, and that these probes can be applied for dynamic studies of highly abundant nuclear RNA. Furthermore, it proved feasible to combine RNA detection with that of green fluorescent protein-labeled proteins in living cells. This was applied to show co-localization of RNA with proteins and should enable RNA–protein interaction studies.     

Long oligonucleotide arrays on nylon for large-scale gene expression analysis

To date, most studies of multigenic expression patterns by long DNA array have used DNA fragments as probes. These probes are usually obtained as PCR products, and this represents a time-consuming and error-prone approach, requiring strict quality control. The present study examines the use of 40- and 70-mer synthetic oligonucleotides as probes for DNA array analysis with radioactive labeled targets. Design, spotting onto nylon filters, and hybridization conditions were determined and optimized. In this approach, the sensitivity and the specificity of the hybridization appear comparable to the conventional long DNA probes assay, permitting the analysis of small samples of approximately 1 microg total RNA. The long oligonucleotide array thus provides a very convenient method for the analysis of gene expression patterns in biological specimens and in clinical research.     

Characterization and application of soybean YACs to molecular cytogenetics

Yeast artificial chromosomes (YACs) are widely used in the physical analysis of complex genomes. In addition to their value in chromosome walking for map-based cloning, YACs represent excellent probes for chromosome mapping using fluorescence in situ hybridization (FISH). We have screened such a library for low-copy-number clones by hybridization to total genomic DNA. Four clones were chosen for chromosome tagging based upon their low or moderate signal. By using degenerate oligonucleotide-primed PCR (DOP-PCR), we were able to use relatively small amounts of soybean YAC DNA, isolated directly by preparative pulsed-field gel electrophoresis, as FISH probes for both metaphase chromosome spreads and interphase nuclei. FISH chromosomal analysis using the three of the clones as probes resulted in relatively simple hybridization patterns consistent with a single homologous locus or two homoeologous loci. The fourth YAC probe resulted in a diffuse hybridization pattern with signal on all metaphase chromosomes. We conclude that YACs represent a valuable source of probes for chromosomal analysis in soybean.     

Discrimination Amongst Leishmania by Polymerase Chain Reaction and Hybridization with Small Subunit Ribosomal DNA Derived Oligonucleotides

ABSTRACT. A method for discriminating among Leishmania is described, based upon small subunit ribosomal DNA sequence differences. The method was to amplify the entire 2.2 kb small subunit rDNA by polymerase chain reaction using conserved primers specific for the 5' and 3' termini of the small subunit ribosomal RNA, and then hybridize the product dotted onto nylon membranes with labeled oligonucleotides. The design of the hybridization probes was based upon complete small subunit rDNA sequences from L. amazonensis, L. major and L. guyanensis and partial sequences of L. mexicana, L. braziliensis, L. tropica and L. chagasi. A high degree of sequence similarity (> 99%) among species was found. However, sufficient sequence divergence occurred to permit the design of internal oligonucleotide probes specific for species complexes. This procedure successfully discriminated amongst a wide range of Leishmania isolates. The method detected as few as 10 cultured organisms and detected parasites in tissue samples from experimentally infected animals. Non-radioactive labeling showed the same specificity and sensitivity as radioactive probes.     

5′-Bispyrene molecular beacons for RNA detection

New fluorescent excimer-forming 5′-bispyrene molecular beacons for the detection of RNA were designed. The probes are 2′-O-methyl RNAs containing 5′-bispyrenylmethylphosphorodiamidate group (bispyrene group) at the 5′-end and a fluorescence quencher (BHQ1) at the 3′-end. A comparative study of the fluorescent properties of the probes having different distance between 5′-bispyrene group and target RNA upon the formation of hybridization complex was performed. The probes with bispyrene group located in the close proximity to the duplex exhibit the greatest excimer fluorescence upon binding to a complementary the 43-nt target RNA, in contrast to the probes with 5′-bispyrene group at dangling end. The feasibility of the new probes for visualization of intracellular RNA was demonstrated using 28S rRNA as a target. The results obtained confirm that the probes proposed in the study can be used as selective tools for RNA detection.