A Sensitive Alternative for MicroRNA In Situ Hybridizations Using Probes of 2��-O-Methyl RNA + LNA

The use of short, high-affinity probes consisting of a combination of DNA and locked nucleic acid (LNA) has enabled the specific detection of microRNAs (miRNAs) by in situ hybridization (ISH). However, detection of low–copy number miRNAs is still not always possible. Here the authors show that probes consisting of 2′-O-methyl RNAs (2OMe) and LNA at every third base (2:1 ratio), under optimized hybridization conditions, excluding yeast RNA from the hybridization buffer, can provide superior performance in detection of miRNA targets in terms of sensitivity and signal-to-noise ratio compared to DNA + LNA probes. Furthermore, they show that hybridizations can be performed in buffers of 4M urea instead of 50% formamide, thereby yielding an equally specific but nontoxic assay. The use of 2OMe + LNA–based probes and the optimized ISH assay enable simple and fast detection of low–copy number miRNA targets, such as miR-130a in mouse brain.     

Single-copy gene detection using branched DNA (bDNA) in situ hybridization.

We have developed a branched DNA in situ hybridization (bDNA ISH) method for detection of human papillomavirus (HPV) DNA in whole cells. Using human cervical cancer cell lines with known copies of HPV DNA, we show that the bDNA ISH method is highly sensitive, detecting as few as one or two copies of HPV DNA per cell. By modifying sample pretreatment, viral mRNA or DNA sequences can be detected using the same set of oligonucleotide probes. In experiments performed on mixed populations of cells, the bDNA ISH method is highly specific and can distinguish cells with HPV-16 from cells with HPV-18 DNA. Furthermore, we demonstrate that the bDNA ISH method provides precise localization, yielding positive signals retained within the subcellular compartments in which the target nucleic acid sequences are localized. As an effective and convenient means for nucleic acid detection, the bDNA ISH method is applicable to the detection of cancers and infectious agents. (J Histochem Cytochem 49:603-611, 2001)     

Improved procedure for the measurement of telomere length in whole cells by PNA probe and flow cytometry

Objectives: Peptide nucleic acid (PNA) probes hybridize to denatured telomeric sequences in cells permeabilized in hot formamide. In reported protocols, the hybridization was conducted in solutions with high formamide concentrations to avoid the DNA renaturation that can hamper binding of the oligo‐PNA probe to specific sequences. We postulated that telomeric DNA, confined in the nuclear microvolume, is not able to properly renature after hot formamide denaturation. Therefore, to improve hybridization conditions between the probe and the target sequences, it might be possible to add probe to sample after the complete removal of formamide. Materials and methods: After telomeric DNA denaturation in hot formamide solution and several washes to remove the ionic solvent, cells were hybridized overnight at room temperature with human telomere‐specific PNA probe conjugated with Cy5 fluorochrome, Cy5‐OO‐(CCCTAA)₃. After stringency washes and staining with ethidium bromide, the cells were analysed by flow cytometry and by using a confocal microscope. Results: Using three continuous cell lines, different in DNA content and telomere length, and resting human peripheral blood T and B lymphocytes, we demonstrated that the oligo‐PNA probe hybridized to telomeric sequences after complete removal of formamide and that in the preserved nucleus, telomeric sequence denaturation is irreversible. Conclusion: According to our experience, oligo‐PNA binding results is efficient, specific and proportional to telomere length. These, our original findings, can form the technological basis of actual in situ hybridization on preserved whole cells.     

Detection of viral infection and gene expression in clinical tissue specimens using branched DNA (bDNA) in situ hybridization.

In situ hybridization (ISH) methods for detection of nucleic acid sequences have proved especially powerful for revealing genetic markers and gene expression in a morphological context. Although target and signal amplification technologies have enabled researchers to detect relatively low-abundance molecules in cell extracts, the sensitive detection of nucleic acid sequences in tissue specimens has proved more challenging. We recently reported the development of a branched DNA (bDNA) ISH method for detection of DNA and mRNA in whole cells. Based on bDNA signal amplification technology, bDNA ISH is highly sensitive and can detect one or two copies of DNA per cell. In this study we evaluated bDNA ISH for detection of nucleic acid sequences in tissue specimens. Using normal and human papillomavirus (HPV)-infected cervical biopsy specimens, we explored the cell type-specific distribution of HPV DNA and mRNA by bDNA ISH. We found that bDNA ISH allowed rapid, sensitive detection of nucleic acids with high specificity while preserving tissue morphology. As an adjunct to conventional histopathology, bDNA ISH may improve diagnostic accuracy and prognosis for viral and neoplastic diseases.     

Influence Factors of in Situ Hybridization in Triticeae

In order to increase the efficiency, accuracy, fidelity and reliability of in situ hybridization to identify the alien chromosomes and chromosome fragments in triticeae, major steps including probe labelling, chromosome denaturation, DNA concentration for blocking and post-hybridization washing in in situ hybridization were optimized. The results are as fel-lows. (1) The cloned repetitive DNA sequence could be biotin labelled more efficiently by nick translation than by random oligonucleotide labelling method: whereas the random oligonucleotide labelling is more suitable for genomic DNA probe and the labelling efficiency could be increased by prolonging the labelling time appropriately. (2) Denaturation of the biotinylated probe and chromosomes together in oven at 75 ℃ showed the satisfactory results of in situ hybridization, but the contour of treated rye chromosomes often became blurred when the temperature of denaturation was higher than 85℃. When 70% formamide (in 2 × SSC) was used to denature the chromosome DNA, rye chromosomes often swelled although the biotinylated signals could be detected. (3) The unlabeled DNA concentrations for blocking were tested in genomic in situ hybridization to detect the Haynaldia villosa chromosomes with biotin labelled H. villosa genomic DNA as probe. The best contrast between H. villosa and wheat chromosomes was obtained without using the blocking DNA (unlabeled wheat genomic DNA). (4) Post-hybridization washes were carried out in 50% formamide (in 2 × SSC) or in 2 × SSC at different temperature. When the post-hybridization washing temperature were increased gradually from room temperature to 42℃ in 50% formamide (in 2 × SSC). specific in situ hybridization signals on chromosome in triticeae were observed using both biotinylated repetitive DNA and genomic DNA as probe. With the improved resolution of this protocol, in situ hybridization would be widely applied to wheat breeding and genetics researches.     

Chromosomal DNA denaturation and reassociation in situ.

The degree of chromosomal DNA (cDNA) denaturation and renaturation on polytene chromosomes has been measured by UV microspectrophotometry. Also DNA losses occurring upon denaturation have been quantified by Feulgen, gallocyanin-chromalum and UV. It has been observed that denaturation in alkali (0.07 N NaOH at room temperature) and formamide (90% formamide; 0.1 SSC, pH 7.2) at 65 °C removes about 30% of the DNA. Low DNA loss occurs upon denaturation in HCl (0.24 M) at room temperature and 60% formamide: 2 × 10^?4 M EDTA (pH 8) at 55 °C. The presence of 4% formaldehyde in the denaturation buffer prevents DNA loss. After denaturation of chromosomes in 0.1 × SSC containing 4% formaldehyde at 100 °C for 30 sec, an hyperchromicity of 39 °C is observed. The denaturation efficiency varies with the denaturation treatment. The percentage reassociation was measured from the difference in the UV absorption of renatured chromosomes and that of denatured chromosomes from the same set. It seems that in our conditions DNA:DNA reassociation does not occur. The efficiency of hybridization is proportional to the denaturation extent of the DNA. However, the entire fraction of DNA which has been denatured is not available for hybridization.     

Detection and amplification systems for sensitive, multiple-target DNA and RNA in situ hybridization: looking inside cells with a spectrum of colors

In situ hybridization (ISH) is a powerful technique for localizing specific nucleic acid sequences (DNA, RNA) in microscopic preparations of tissues, cells, chromosomes, and linear DNA fibers. To date, a wide variety of research and diagnostic applications of ISH have been described, making the technique an integral part of studies concerning gene mapping, gene expression, RNA processing and transport, the three-dimensional organization of the nucleus, tumor genetics, microbial infections, and prenatal diagnosis. In this review, I first describe the ISH procedure in short and then focus on the currently available non-radioactive probe-labeling and cytochemical detection methodologies that are utilized to visualize one or multiple different nucleic acid targets in situ with different colors. Special emphasis is placed on the procedures applying fluorescence and brightfield microscopy, the simultaneous detection of nucleic acids and proteins by combined ISH and immunocytochemistry, and, in addition, on the recent progress that has been made with the introduction of signal amplification procedures to increase the detection sensitivity of ISH. Finally, a comparison of fluorescence, enzyme cytochemical, and colloidal gold silver probe detection systems will be presented, and possible future directions of in situ nucleic acid detection will be discussed. Accepted: 9 June 1999     

Denaturation, renaturation, and loss of DNA during in situ hybridization procedures

With the aim of optimizing in situ hybridization methods, alkaline, acid, and thermal denaturation procedures have been studied for their ability to separate the DNA strands of nuclear DNA and for the DNA losses they induce. Isolated methanol/acetic acid-fixed mouse liver nuclei have been used as a biological object. The results, obtained with acridine orange staining and microfluorometry, show that all denaturations studied lead to almost complete strand separation. Quantitative DNA staining and cytometry indicated that with heat and alkaline denaturation about 40% of the DNA is lost. Acid denaturation led to about 20% DNA loss. For the alkaline denaturation, the DNA retention could be improved to a 20% DNA loss by adding 70% ethanol to the denaturation medium. During hybridization, another 20% DNA loss occurs. When denatured nuclei are brought under annealing conditions, a rapid renaturation of a considerable fraction of the remaining DNA occurs. The extent of renaturation was dependent on the type of denaturation used. For the ethanolic alkaline denaturation, it was estimated to be 35%. Quantitative nonautoradiographic in situ hybridization experiments with acetylaminofluorene-modified mouse satellite DNA showed that alkaline denaturation procedures are superior to the heat and acid denaturation. As proven by acridine orange fluorescence measurements, hybridization conditions can be designed that permit DNA.RNA hybridization under in situ DNA.DNA denaturing conditions. These conditions should be very useful, especially for in situ hybridization with single-stranded RNA probes.     

Optimization of the conditions for the in situ hybridization of cloned DNA sequences and for the differential staining of human chromosomes

The influence of different experimental conditions on in situ hybridization of DNA and subsequent differential staining of chromosomes was studied. The most optimal conditions for chromosomal localization of cloned repetitive DNA sequences were the lack of chromosome pretreatment with acid and RNase, reduction of the denaturation time to 30 s, carrying out of hybridization at a relatively low temperature (under 37 degrees C) at the expense of the use of formamide, addition to the hybridization mixture of 10% of dextran sulfate-500. The conditions indicated permit obtaining on radioautographs the G- and C-segmentation of human chromosomes.     

Surface plasmon field-enhanced fluorescence spectroscopy in PCR product analysis by peptide nucleic acid probes

Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) was recently developed for PCR product analysis, which allowed for real-time monitoring of hybridization processes and for the detection of trace amounts of PCR products, with a detection limit of 100 fmol on the peptide nucleic acid (PNA) probe surface, and 500 fmol on the DNA probe surface. By selectively labeling the strands of PCR-amplified DNA, it was shown that the heat denaturation process in combination with the application of low-salt condition substantially reduced the interference from the antisense strands and thus simplified the surface hybridization. Furthermore, SPFS was demonstrated to be capable of quantitatively discriminating the difference induced by single nucleotide substitution, even within one minute of contact time.     

Amplification methods to increase the sensitivity of in situ hybridization: play card(s).

In situ hybridization (ISH) has proved to be an invaluable molecular tool in research and diagnosis to visualize nucleic acids in their cellular environment. However, its applicability can be limited by its restricted detection sensitivity. During the past 10 years, several strategies have been developed to improve the threshold levels of nucleic acid detection in situ by amplification of either target nucleic acid sequences before ISH (e.g., in situ PCR) or the detection signals after the hybridization procedures. Here we outline the principles of tyramide signal amplification using the catalyzed reporter deposition (CARD) technique, present practical suggestions to efficiently enhance the sensitivity of ISH with CARD, and discuss some applications and possible future directions of in situ nucleic acid detection using such an amplification strategy.     

ISH-guided freeze-matrix assisted punches: technique for extracting punches from thin slide-mounted tissues for DNA methylation analysis

Dissection of discrete brain regions for molecular analysis is complicated by trade-offs between accuracy, flexibility, and costs. We developed a flexible and cost-effective method, in situ hybridization (ISH) guided freeze-matrix assisted punches (IFAP), for extracting nanogram quantities of DNA from slide-mounted sections as thin as 12 μm. Using ISH to localize regions of interest, tissue is targeted by applying a small bead of M-1 embedding matrix onto cryosections, snap-freezing, and collecting the beads for nucleic acid purification. The method quantitatively recovers RNA and DNA usable for PCR and DNA methylation analysis.     

Nucleic acid hybridization using DNA covalently coupled to cellulose.

We describe a method for linking RNA and DNA covalently to finely divided cellulose through a diazotized aryl amine, which reacts primarily with guanine and uracil (thymine) residues of single strands. The high efficiency of coupling and high capacity of the cellulose for nucleic acid make possible a product with as much as 67 mug of nucleic acid per mg of cellulose. The product is especially suitable for hybridization experiments where very low backgrounds are important, and it is stable in 99% formamide at 80 degrees C so that hybridized nucleic acid can be recovered easily. Full length linear Simian Virus 40 (SV40) DNA, produced by cleavage of SV40(I) DNA with S1 nuclease, can be coupled to diazo cellulose with an efficiency of 80-90%, and is effective in hybridization experiments with SV40 DNA, complementary RNA synthesized in vitro from SV40(I) DNA with E. coli RNA polymerase, and the SV40-specific fraction of total RNA from SV40-infected and transformed cells. In these experiments an excess of cellulose-bound DNA was used, and the efficiency of hybridization was about 90% when ribonuclease treatment of the hybrids was omitted.     

Influence of secondary structure on kinetics and reaction mechanism of DNA hybridization

Hybridization of nucleic acids with secondary structure is involved in many biological processes and technological applications. To gain more insight into its mechanism, we have investigated the kinetics of DNA hybridization/denaturation via fluorescence resonance energy transfer (FRET) on perfectly matched and single-base-mismatched DNA strands. DNA hybridization shows non-Arrhenius behavior. At high temperature, the apparent activation energies of DNA hybridization are negative and independent of secondary structure. In contrast, when temperature decreases, the apparent activation energies of DNA hybridization change to positive and become structure dependent. The large unfavorable enthalpy of secondary structure melting is compensated for by concomitant duplex formation. Based on our results, we propose a reaction mechanism about how the melting of secondary structure influences the hybridization process. A significant point in the mechanism is that the rate-limiting step switches along with temperature variation in the hybridization process of structured DNA, because the free energy profile of hybridization in structured DNA varies with the variation in temperature.     

RNA:DNA hybrids are more stable than DNA:DNA duplexes in concentrated perchlorate and trichloroacetate solutions.

Rates of formation of RNA:DNA hybrids have been measured as a function of temperature and compared to DNA:RNA duplex denaturation temperatures in 4 M sodium perchlorate, 4 M NaClO4-6 M urea, and 3 M rubidium trichloracetate solvents. The usual bell shaped curves of reaction rate versus temperature were observed. The optimal temperatures for the RNA:DNA association reaction are 5 degrees to 12 degrees greater than the Tm's for DNA:DNA denaturation in these solvents, just as in formamide. R-loops of phi80d3ilv DNA with E. coli rRNA can be formed at high efficiency in these solvents.     

ChipCheckII - predicting binding curves for multiple analyte strands on small DNA microarrays

Incomplete binding, saturation, and cross-hybridization between partially complementary strands complicate the parallel detection of nucleic acids via DNA microarrays. Treating the competing equilibria governing binding to microarrays requires computational tools. We have developed the web-based program ChipCheckII that calculates total hybridization matrices for target strands interacting with probes on small DNA microarrays. The program can be used to compute the extent of cross-hybridization and other phenomena affecting fidelity of detection based on sequences, quantities of strands, and hybridization conditions as inputs. Enthalpy and entropy of duplex formation are generated locally with UNAfold, including those for complexes that are partially matched. Simulated binding versus temperature curves for portions of a commercial genome chip demonstrate the extent to which cross-hybridization can complicate DNA detection. ChipCheckII is expected to aid nucleic acid chemists in developing high fidelity DNA microarrays.     

ChipcheckII—Predicting Binding Curves for Multiple Analyte Strands on Small DNA Microarrays

Incomplete binding, saturation, and cross-hybridization between partially complementary strands complicate the parallel detection of nucleic acids via DNA microarrays. Treating the competing equilibria governing binding to microarrays requires computational tools. We have developed the web-based program ChipCheckII that calculates total hybridization matrices for target strands interacting with probes on small DNA microarrays. The program can be used to compute the extent of cross-hybridization and other phenomena affecting fidelity of detection based on sequences, quantities of strands, and hybridization conditions as inputs. Enthalpy and entropy of duplex formation are generated locally with UNAfold, including those for complexes that are partially matched. Simulated binding versus temperature curves for portions of a commercial genome chip demonstrate the extent to which cross-hybridization can complicate DNA detection. ChipCheckII is expected to aid nucleic acid chemists in developing high fidelity DNA microarrays.     

Simultaneous Detection of FISH Signals and Bromo-Deoxyuridine Incorporation in Fixed Tissue Cultured Cells

FISH (Fluorescence in situ hybridization) is a powerful technique that detects and localises specific DNA sequences on metaphase chromosomes, interphase nuclei or chromatin fibres. When coupled to BrdU (5-Bromo 2-deoxy-uridine) labeling of newly replicated DNA, the replication properties of different DNA sequences can be analysed. However, the technique for the detection of BrdU incorporation is time consuming, and relies on acidic pH buffer treatments, that prevent use of pH sensitive fluorochromes such as FITC (Fluoro-isothiocianate) during FISH. In this work, we describe a simplified protocol that allows the simultaneous detection of FISH signals and BrdU incorporation. Since the technique does not involve paraformaldehyde for cell fixation, or formamide for denaturation of the target DNA and in post-hybridisation washes, it represents a safer alternative to classical FISH techniques.     

Co-labeling using in situ PCR: a review.

In situ amplification permits the histological localization of low-copy DNA and RNA targets. However, in many instances it would be useful to know the specific phenotype of the target-containing cell or to ascertain the distribution of a different nucleic acid sequence in the same tissue section. This review describes a methodology that allows co-in situ localization of two nucleic acid targets or a DNA/RNA sequence and a protein in paraffin-embedded, formalin-fixed tissue. The key variable for detection of low-copy RNA targets by RT in situ PCR is optimal protease digestion to permit cDNA target-specific incorporation of the reporter nucleotide. This is achieved via inactivation of nonspecific DNA synthesis by overnight DNase digestion. The key variable for immunohistochemical localization of proteins is to determine the effect of protease digestion on the antigen-based signal intensity. Background for DNA targets by in situ hybridization or, for targets present in 1-10 copies per cell, PCR ISH is dependent primarily on probe concentration and the stringency of the post-hybridization wash. Radioactive 3H-labeled nucleotides permit an excellent distinction with colorimetric signals for co-localization, although two distinct chromogens can in many instances allow successful localization of two different targets.