A new case of neuropeptide coexpression (RGamide and LWamides) in Hydra,found by whole-mount,two-color double-labeling in situ hybridization

The freshwater polyp Hydra has a primitive nervous system that expresses at least six different neuropeptide genes: (1) three genes, coding for the preprohormones-A, -B, and -C that each gives rise to a variety of peptides with the C-terminal sequence Arg-Phe-NH(2) (the Hydra-RFamides); (2) one gene, coding for a preprohormone that gives rise to five peptides with the C-terminal sequence Leu-Trp-NH(2) (the Hydra-LWamides); (3) one gene, coding for a preprohormone that produces a peptide with the C-terminal sequence Lys-Val-NH(2) (Hydra-KVamide, also called Hym-176); and (4) one gene, coding for a preprohormone that gives rise to a peptide with the C-terminal sequence Arg-Gly NH(2) (Hydra-RGamide, also called Hym-355). In a previous paper, we described that a population of neurons in the peduncle (a region just above the foot) of Hydra coexpresses the preprohormone-A and KVamide genes, whereas neurons in the other regions only express either the preprohormone-A, -B, -C, LWamide, or the KVamide genes. Here, we investigated the RGamide gene expression, using whole-mount, two-color double-labeling in situ hybridization, and found that neurons in the basal disk (foot), gastric region, hypostome (a region around the mouth), and tentacles coexpress this gene together with the LWamide gene. A small population of neurons in the hypostome and upper gastric region expresses only the LWamide gene. No coexpression of the RGamide gene with any of the other neuropeptide genes was observed. This is the second example of coexpression of two neuropeptide genes in cnidarians. It demonstrates that many neurons in the primitive nervous systems of cnidarians use combinations ("cocktails") of neuropeptides for their signaling. It also shows that Hydra has at least seven neurochemically different populations of neurons.     

Dramatically improved RNA in situ hybridization signals using LNA-modified probes

In situ detection of RNA by hybridization with complementary probes is a powerful technique. Probe design is a critical parameter in successful target detection. We have evaluated the efficiency of fluorescent DNA oligonucleotides modified to contain locked nucleic acid (LNA) residues. This increases the thermal stability of hybrids formed with RNA. The LNA-based probes detect specific RNAs in fixed yeast cells with an efficiency far better than conventional DNA oligonucleotide probes of the same sequence. Using this probe design, we were also able to detect poly(A)(+) RNA accumulation within the nucleus/ nucleolus of wild-type cells. LNA-based probes should be readily applicable to a diverse array of cells and tissue samples.     

In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using antisense RNA probes

In situ hybridization studies with [32P] and [3H] labelled antisense RNA probes were undertaken to determine optimal methods of tissue fixation, tissue sectioning, and conditions of hybridization, and to compare the relative merits of the two different radioactive labels. The distribution of somatostatin mRNA in neurons of rat brain using a labelled antisense somatostatin RNA probe was employed as a model for these studies. The highest degree of sensitivity for in situ hybridization was obtained using paraformaldehyde fixation and vibratome sectioning. Optimal autoradiographic localization of mRNA was obtained within 7 days using [32P] labelled probes. However, due to the high energy emittance of [32P], precise intracellular localization of hybridization sites was not possible. [3H] labelled RNA probes gave more precise cellular localization but required an average of 18-20 days autoradiographic exposure. The addition of the scintillator, PPO, decreased the exposure time for the localization of [3H] labelled probes to seven days. We also report a method for combined in situ hybridization and immunocytochemistry for the simultaneous localization of somatostatin in mRNA and peptide in individual neurons.     

Hybridizationin situ of whole-mount messenger RNA in plants

A procedure is presented for the detection of mRNA in whole-mount preparations of youngArabidopsis seedlings using digoxigenin (DIG)-labeled RNA probes. It includes tissue preservation with formaldehyde, permeabilization with polyoxyethy lenesorbitan (Tween 20), DMSO, and heptane. Hybridization signal is detected using colloidal gold or alkaline-phosphatase-conjugated anti-DIG antibodies.     

PCR-derived ssDNA probes for fluorescent in situ hybridization to HIV-1 RNA.

We developed a simple and rapid technique to synthesize single-stranded DNA (ssDNA) probes for fluorescent in situ hybridization (ISH) to human immunodeficiency virus 1 (HIV-1) RNA. The target HIV-1 regions were amplified by the polymerase chain reaction (PCR) and were simultaneously labeled with dUTP. This product served as template for an optimized asymmetric PCR (one-primer PCR) that incorporated digoxigenin (dig)-labeled dUTP. The input DNA was subsequently digested by uracil DNA glycosylase, leaving intact, single-stranded, digoxigenin-labeled DNA probe. A cocktail of ssDNA probes representing 55% of the HIV-1 genome was hybridized to HIV-1-infected 8E5 T-cells and uninfected H9 T-cells. For comparison, parallel hybridizations were done with a plasmid-derived RNA probe mix covering 85% of the genome and a PCR-derived RNA probe mix covering 63% of the genome. All three probe types produced bright signals, but the best signal-to-noise ratios and the highest sensitivities were obtained with the ssDNA probe. In addition, the ssDNA probe syntheses generated large amounts of probe (0.5 to 1 microg ssDNA probe per synthesis) and were easier to perform than the RNA probe syntheses. These results suggest that ssDNA probes may be preferable to RNA probes for fluorescent ISH. (J Histochem Cytochem 48:285-293, 2000)     

Specific distribution of mRNAs in maize growing pollen tubes observed by whole-mount in situ hybridization with non-radioactive probes

The distribution of a number of specific mRNAs has been observed in maize growing pollen tubes. Whole-mount in situ hybridization using digoxygenin-labelled RNA probes has been tested. The technique appears to be a simple, rapid and reliable method in this system, in immature anthers and also in other tissues. Results for three probes are presented. They correspond to a hydroxyproline-rich glycoprotein (HRGP), an abundant component of the maize cell wall, to an α-tubulin (encoded by the Tubα 1 gene) highly expressed in the radicular system of the plant and also in pollen, and to an isoform of the malic enzyme, involved in the basic metabolism of the plant. The mRNAs corresponding to these three proteins are differently distributed in the germinating pollen. While HRGP mRNA is only present in the tube, malic enzyme mRNA is only present in the body of the pollen cell, and α-tubulin mRNA is present in both parts of the cell but shows a higher accumulation in the tip of the pollen tube.     

Preparation of nonradioactive probes for in situ hybridization

In situ hybridization (ISH) enables the precise localization of RNA targets and provides an avenue to study the temporal and spatial patterns of expression of specific genes. ISH has evolved from being an esoteric technique to one that is routinely used by researchers in many areas of research. A major driving force has been the development of numerous nonisotopic labeling and signal detection methods. Historically, radioactive probes and autoradiography provided sensitivity that was unattainable with nonisotopic probes. But the long exposure times required for signal detection and the perceived dangers associated with radioactivity limit its use. Advances in nonisotopic detection systems have overcome many of the limitations associated with using radiolabeled probes. One of the most significant contributions from nonisotopic methods is the ability to discriminate between multiple nucleic acid sequences simultaneously.     

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.     

Specific detection of Pseudomonas spp. in milk by fluorescence in situ hybridization using ribosomal RNA directed probes

AIMS: Pseudomonas spp. are considered the most important milk spoilage organisms. Here we describe development of a fluorescence in situ hybridization (FISH) probe specific for detection and enumeration of Pseudomonas spp. in milk. METHODS AND RESULTS: 16S rRNA sequences were analysed to develop specific oligonucleotide probe for the genus Pseudomonas. Twenty different Pseudomonas spp. and 23 bacterial species from genera other than Pseudomonas (as negative controls) were tested. All tested Pseudomonas spp. yielded a positive FISH reaction, whereas negative controls showed no FISH reaction except for Burkholderia cepacia that showed a relatively weak FISH reaction. The FISH assay specifically stains Pseudomonas in milk when the milk contains a mixture of other bacterial species. The FISH assay takes 2 h and compares favourably with current culturing methods, which take a minimum of 48 h. Specificity of the probe was validated using polymerase chain reaction to selectively amplifying the Pseudomonas rDNA gene and sequencing the gene products. CONCLUSIONS: The method presented in this study allows simultaneously detection, identification and enumeration of Pseudomonas spp. in milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid and accurate enumeration of Pseudomonas facilitates the identification of specific contamination sources in dairy plants, the accurate validation of pasteurization treatments and the prediction of shelf life of processed milk.     

A nonradioactive whole-mount in situ hybridization protocol for Porphyra (Rhodophyta) gametophytic germlings

The objective of this study was to develop a method for whole-mount in situ hybridization (WISH) by using elongation factor-1 (EF-1) riboprobes in the red alga Porphyra yezoensis Ueda. Several modifications to the general WISH protocol, such as use of a short-length probe, performing partial digestion of the cell wall, optimization of proteinase K concentration and additional washing steps after hybridization were essential to reduce non-specific staining and to obtain sufficient quality of data. This protocol made it possible to detect a specific signal as a positive control in WISH assays of P. yezoensis.     

Mapping muscle protein genes by in situ hybridization using biotin-labeled probes.

The genes coding for the myosin heavy chain isoforms (unc-54, myo-1, myo-2 and myo-3) and the actins (act-1,2,3 and act-4) have been mapped on the embryonic metaphase chromosomes of Caenorhabditis elegans by in situ hybridization. The genes were cloned in a cosmid vector and the entire cosmid was nick translated to incorporate biotin-labeled dUTP. This produced a probe DNA complementary to a 35-45 kb length of chromosomal DNA. The hybridization signal from the cosmid probe, detected by immunofluorescence, could be easily seen by eye. The clear signals and the specific hybridization of the cosmid probes provided a faster means of mapping these single copy genes than small probes cloned in plasmid or lambda vectors. The myosin heavy chain genes are not clustered. Only unc-54 and myo-1 mapped to the same chromosome; the unc-54 locus is at the extreme right end of linkage group I and myo-1 mapped 40-50% from the left end of linkage group I. Myo-2 mapped to the X, 52-75% from the left end. The myo-3 gene mapped to the middle of linkage group V near the cluster of three actin genes (act-1,2,3). The fourth actin gene, act-4 mapped to 20-35% from the left end of X.     

An optimized procedure for whole-mount in situ hybridization on mouse embryos and embryoid bodies

Determining the precise expression pattern of a gene of interest at various stages of development is essential to understanding its biological function in embryology. This protocol describes a sensitive method for whole-mount in situ hybridization (WISH) to mouse embryos, using cRNA probes. Adaptations are provided that allow the protocol to be applied to embryonic stages ranging from blastocysts to postimplantation stage embryos, and to embryoid bodies. We also describe an in situ method for differential detection of two probes. Probe labeling and dissection and preparation of the embryos can be performed in 2 d. The actual WISH procedure can be completed in another 3 d.     

Construction of repeat-free fluorescence in situ hybridization probes

FISH probes are generally made out of BAC clones with genomic DNA containing a variable amount of repetitive DNA that will need to be removed or blocked for FISH analysis. To generate repeat free (RF) Probes without loss in genomic coverage, a random library is made from BAC clones by whole-genome amplification (WGA). Libraries are denatured in the presence of excess C(0)t-1 DNA and allowed to re-anneal followed by digestion of all double-stranded elements by duplex-specific nuclease (DSN). Selective amplification of all elements not containing repetitive sequences is realized by a sequential amplification. The final RF products can be re-amplified and used as a stock for future probe production. The RF probes have a lower background, the signal intensity build up is faster and there is no need for blocking DNA. The signal to background ratio of the RF was higher as compared to repeat containing probes.