A biotinylated DNA probe to detect bacterial cells in artificially contaminated foodstuffs

A biotin-labelled DNA probe was used in a dot-blot hybridization test to demonstrate the presence of Escherichia coli in a variety of artificially contaminated foodstuffs. Positive hybridization was detected by using a streptavidine/polyalkaline phosphatase conjugate to generate an insoluble coloured precipitate in the presence of an appropriate dye. The colour intensity was measured with a computer-controlled image analysis system which assessed objectively the hybridization signal produced by each sample. The method was capable of distinguishing positive hybridization at cell concentrations exceeding 10(4) cells/dot-blot, equivalent to 2 x 10(7) cells/g food, and had none of the drawbacks normally associated with the use of radioactively labelled DNA in hybridization techniques. The procedure is highly specific and takes less than 30 h. Many samples can be screened simultaneously and the procedure can be used to detect any species for which a suitable DNA probe is available.     

A biotinylated DNA probe to detect bacterial cells in artificially contaminated foodstuffs

A biotin-labelled DNA probe was used in a dot-blot hybridization test to demonstrate the presence of Escherichia coli in a variety of artificially contaminated foodstuffs. Positive hybridization was detected by using a streptavidine/polyalkaline phosphatase conjugate to generate an insoluble coloured precipitate in the presence of an appropriate dye. The colour intensity was measured with a computer-controlled image analysis system which assessed objectively the hybridization signal produced by each sample. The method was capable of distinguishing positive hybridization at cell concentrations exceeding 10⁴ cells/dot-blot, equivalent to 2×10⁷ cells/g food, and had none of the drawbacks normally associated with the use of radioactively labelled DNA in hybridization techniques. The procedure is highly specific and takes less than 30 h. Many samples can be screened simultaneously and the procedure can be used to detect any species for which a suitable DNA probe is available     

Ribavirin cures cells of a persistent infection with foot-and-mouth disease virus in vitro.

Ribavirin (1-beta-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide) eliminates foot-and-mouth disease virus from persistently infected cell cultures. The latter are 10-fold more sensitive to ribavirin than lytically infected cells. In treated cells no viral RNA or proteins could be detected by dot-blot hybridization to cDNA probes, virus and RNA infectivity assays, or immunofluorescence. A potential application of the drug for the treatment of animals carrying the virus is suggested.     

An alternative nonradioactive method for labeling DNA using biotin

An alternative nonradioactive labeling method and a highly sensitive technique for detecting specific DNA sequences are described. The labeling method requires the "Klenow" fragment of DNA polymerase I and random hexanucleotides (synthesized or naturally extracted) as a primer for the production of highly sensitive DNA probes. The system has three main steps: (i) labeling of DNA with biotinylated 11-dUTP; (ii) detection of biotinylated DNA by a one-step procedure with streptavidin-alkaline phosphatase complex; (iii) blocking of background with Tween 20. Twenty attograms (2 X 10(-17) g) of pBR322 plasmid DNA was detected by dot-blot hybridization. Upon Southern blot hybridization, 7.4 fg (7.4 X 10(-15) g) of pBR322 HindIII DNA was detected using the biotinylated pBR322 plasmid DNA probe; 40.8 ag and 7.4 fg of lambda HindIII DNA were detected with the biotinylated whole lambda DNA probe by dot and Southern blot hybridization, respectively. Specific bands were also detected with the biotinylated argininosuccinolyase probe upon Northern blotting of mouse poly(A+) RNA. Further applications for in situ hybridization are also described.     

Highly sensitive fluorescent-labeled probes and glass slide hybridization for the detection of plant RNA viruses and a viroid

In this study, a modified method of the conventional RNA dot-blot hybridization was established, by replacing ~(32)p labels with CY5 labels and replacing nylon membranes with positive-charged glass slides, for detecting plant RNA viruses and a viroid. The modified RNA dot-blot hybridization method was named glass slide hybridization. The optimum efficiency of RNA binding onto the surfaces of activated glass slide was achieved using aminosilane-coated glass slide as a solid matrix and 5×saline sodium citrate (SSC) as a spotting solution. Using a CY5-labeled DNA probe prepared through PCR amplification, the optimized glass slide hybridization could detect as little as 1.71 pg of tobacco mosaic virus (TMV) RNA. The sensitivity of the modified method was four times that of dot-blot hybridization on nylon membrane with a ~(32)P-labeled probe. The absence of false positive within the genus Potyvirus [potato virus A, potato virus Y (PVY) and zucchini yellow mosaic virus] showed that this method was highly specific. Furthermore, potato spindle tuber viroid (PSTVd) was also detected specifically. A test of 40 field potato samples showed that this method was equivalent to the conventional dot-blot hybridization for detecting PVY and PSTVd. To our knowledge, this is the first report of using dot-blot hybridization on glass slides with fluorescent-labeled probes for detecting plant RNA viruses and a viroid.     

The search for an optimal DNA, RNA, and protein detection by in situ hybridization, immunohistochemistry, and solution-based methods

Clinical trials and correlative laboratory research are increasingly reliant upon archived paraffin-embedded samples. Therefore, the proper processing of biological samples is an important step to sample preservation and for downstream analyses like the detection of a wide variety of targets including micro RNA, DNA and proteins. This paper analyzed the question whether routine fixation of cells and tissues in 10% buffered formalin is optimal for in situ and solution phase analyses by comparing this fixative to a variety of cross linking and alcohol (denaturing) fixatives. We examined the ability of nine commonly used fixative regimens to preserve cell morphology and DNA/RNA/protein quality for these applications. Epstein-Barr virus (EBV) and bovine papillomavirus (BPV)-infected tissues and cells were used as our model systems. Our evaluation showed that the optimal fixative in cell preparations for molecular hybridization techniques was "gentle" fixative with a cross-linker such as paraformaldehyde or a short incubation in 10% buffered formalin. The optimal fixatives for tissue were either paraformaldehyde or low concentration of formalin (5% of formalin). Methanol was the best of the non cross-linking fixatives for in situ hybridization and immunohistochemistry. For PCR-based detection of DNA or RNA, some denaturing fixatives like acetone and methanol as well as "gentle" cross-linking fixatives like paraformaldehyde out-performed other fixatives. Long term fixation was not proposed for DNA/RNA-based assays. The typical long-term fixation of cells and tissues in 10% buffered formalin is not optimal for combined analyses by in situ hybridization, immunohistochemistry, or--if one does not have unfixed tissues--solution phase PCR. Rather, we recommend short term less intense cross linking fixation if one wishes to use the same cells/tissue for in situ hybridization, immunohistochemistry, and solution phase PCR.     

Isolation of RNA for dot hybridization by heparin-DNase I treatment of whole cell lysate

We have developed a new procedure for the rapid preparation of undegraded total RNA from cultured cells for specific quantitation by dot blotting analysis. Pelleted cells are resuspended in hypotonic solution containing a ribonuclease inhibitor and heparin and disrupted by freeze-thaw. Heparin is employed as an agent for nuclear lysis, dissociation of chromosomal protein, and release of mRNA from rough endoplasmic reticulum. We eliminate chromosomal DNA by digestion with DNase I and denature the RNA in the lysate with formaldehyde. After centrifugation to remove debris, the supernatant is used directly for dot blotting. All manipulations are performed in the same microfuge tube and recovery of RNA is quantitative. The procedure is especially useful for processing large numbers of samples. We illustrate its versatility by analysis of specific RNAs in Drosophila, rat, and human cell lines. In reconstruction experiments, less than 80 molecules per cell of a small RNA (beta-globin) can be detected under highly stringent hybridization conditions, using only moderately labeled double-stranded plasmid DNA probes and short film exposures.     

Dot-blot hybridization and RT-PCR detection of extra small virus (XSV) associated with white tail disease of prawn Macrobrachium rosenbergii

The availability of specific and reliable detection methods is essential for monitoring the health status of farmed species, particularly for viral diseases. Extra small virus (XSV), a virus-like particle, is associated with Macrobrachium rosenbergii Noda virus (MrNV) in white tail disease (WTD) of M. rosenbergii. We developed 2 genome-based detection methods for the identification of XSV, namely dot-blot hybridization and a single-step RT-PCR. Detection limits were established and are ca. 2.5 pg and 5 fg of viral RNA for dot-blot hybridization and RT-PCR, respectively. Application of the methods to field samples indicated that some animals positively diagnosed with MrNV did not contain XSV, at least within the detection limit of the methodology. This raises the question of the actual role of XSV and its interactions with MrNV in WTD of M. rosenbergii.     

A total extract dot blot hybridization procedure for mRNA quantitation in small samples of tissues or cultured cells

A simple method for the estimation of specific mRNA concentrations in small tissue samples (as little as 1 mg) or cultured cells (lower limit 10(5) cells) is described. Guanidine hydrochloride extracts of whole cells or tissues are applied directly onto nitrocellulose and hybridized with the appropriate nick-translated probe. Loading according to DNA content allows expression of the result as concentration per cell. Hybridizing with a ribosomal RNA probe allows expression of results relative to rRNA and estimation of the RNA/DNA ratio in the sample. We describe the application of this procedure to the measurement of ceruloplasmin mRNA in tissues and cultured hepatocytes.     

Evidence for a partial RNA transcript of the small circular component of kinetoplast DNA of Crithidia acanthocephali.

The major component of kinetoplast DNA (kDNA) in the protozoan Crithidia acanthocephali is an association of approximately 27,000, 0.8 micrometers (1.58 x 10(6) dalton) circular molecules apparently held together in a particular structural configuration by topological interlocking. We have carried out hybridization experiments between kDNA samples containing one or the other of the two complementary (H and L) strands of purified 0.8 micrometers molecules derived from mechanically disrupted associations and RNA samples prepared either from whole C. acanthocephali cells or from a mitochondrion-enriched fraction. The results of experiments involving cesium sulfate buoyant density centrifugation indicate that whole cell RNA contains a component(s) complementary to all kDNA H strands, but none complementary to kDNA L strands. Similar results were obtained using mitochondrion-associated RNA. Digestion of RNA/DNA hybrids and suitable controls with the single-strand-specific nuclease S1 indicated that 10% of the kDNA H strand is involved in hybrid formation. Visualization of RNA/DNA hybrids stained with bacteriophage T4 gene 32 protein revealed that hybridation involves a single region of each kDNA H strand, equal to approximately 10% of the molecule length. These data suggest that at least 10% of the small circular component of kDNA of Crithidia acanthocephali is transcribed.     

Microbial Composition and Structure of a Rotating Biological Contactor Biofilm Treating Ammonium-Rich Wastewater without Organic Carbon

High nitrogen losses were observed in a rotating biological contactor (RBC) treating ammonium-rich (up to 500 mg NH4(+)-N/L) but organic-carbon-poor leachate from a hazardous waste landfill in K?lliken, Switzerland. The composition and spatial structure of the microbial community in the biofilm on the RBC was analyzed with specific attention for the presence of aerobic ammonium and nitrite oxidizing bacteria and anaerobic ammonium oxidizers. Anaerobic ammonium oxidation (anammox) involves the oxidation of ammonium with nitrite to N2. First the diversity of the biofilm community was determined from sequencing cloned PCR-amplified 16S rDNA fragments. This revealed the presence of a number of very unusual 16S rDNA sequences, but very few sequences related to known ammonium or nitrite oxidizing bacteria. From analysis of biofilm samples by fluorescence in situ hybridization with known phylogenetic probes and by dot-blot hybridization of the same probes to total RNA purified from biofilm samples, the main groups of microorganisms constituting the biofilm were found to be ammonium-oxidizing bacteria from the Nitrosomonas europaea/eutropha group, anaerobic ammonium-oxidizing bacteria of the "Candidatus Kuenenia stuttgartiensis" type, filamentous bacteria from the phylum Bacteroidetes, and nitrite-oxidizing bacteria from the genus Nitrospira. Aerobic and anaerobic ammonium-oxidizing bacteria were present in similar amounts of around 20 to 30% of the biomass, whereas members of the CFB phylum were present at around 7%. Nitrite oxidizing bacteria were only present in relatively low amounts (less than 5% determined with fluorescence in situ hybridization). Data from 16S rRNA dot-blot and in situ hybridization were not in all cases congruent. FISH analysis of thin-sliced and fixed biofilm samples clearly showed that the aerobic nitrifiers were located at the top of the biofilm in an extremely high density and in alternating clusters. Anammox bacteria were exclusively present in the lower half of the biofilm, whereas CFB-type filamentous bacteria were present throughout the biofilm. The structure and composition of these biofilms correlated very nicely with the proposed physiological functional separations in ammonium conversion.     

SV40 RNA: Filter hybridization for rapid isolation and characterization of rare RNAs

Modifications of the filter hybridization method for the measurement and isolation of simian virus 40 (SV40) RNA from transformed cells are described. The modified method used small (0.02 cm²) nitrocellulose filters with > 30 μg/cm² SV40 DNA applied following formaldehyde denaturation. The small volume and high DNA densities allowed hybridization to be completed in 2 h and washing after hybridization to be completed in 6 h. The washing reduced background to 1 × 10^?5 of input radioactivity without using nucleases. The efficiency of hybridization after washing was 40% or greater. These procedures have allowed the quantification of proportions of SV40 RNA in labeled RNA from transformed lines and the characterization of SV40 RNAs by electrophoresis and cell-free translation. A 3.7-kb SV40 RNA from SV80 cells was discovered in this work.     

Determination of tissue-type plasminogen-activator mRNA in human and non-human cell lines by dot-blot hybridization.

A labelled cDNA clone was used in DNA-RNA hybridization on nitrocellulose filter paper (dot-blot technique) to detect and quantify mRNA for endogenous tissue plasminogen activator (PA) in cell extracts and samples of RNA purification runs. Although, for detection purposes, the assay was less sensitive than translation in Xenopus oocytes, it was at least as reliable and much more convenient for the purpose of quantitative determination. In particular, the technique was used to study the kinetics of PA mRNA formation in a human melanoma cell line (Bowes) after exposure to the tumour promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). Incubation of the cells with TPA resulted in a 15-20-fold increase in cellular PA mRNA content. The effect was time- and dose-dependent: the increase in PA-specific mRNA was clearly visible as early as 4 h after initiation of TPA treatment in Bowes cells. It was blocked completely by pretreatment of the cells with actinomycin D, indicating that TPA caused enhancement of synthesis of PA mRNA rather than inhibition of PA mRNA degradation. The use of the nitrocellulose dot-blot technique also revealed that two non-human cell lines produce mRNAs which cross-react with the human PA mRNA, namely the mouse melanoma cell line B16 and the rat brain-tumour cell line, RT4-71-1. TPA was found to exert similar stimulatory effects on the synthesis of mRNAs in these cell lines as in Bowes cells.     

A noise-free molecular hybridization procedure for measuring RNA in cell lysates

A solution hybridization technique was designed to measure RNA abundance in crude cell lysates and at the same time to maximize confidence that signals resulted from true molecular hybridization. Cell lysates were prepared in 5 M guanidine thiocyanate, then RNA molecules in the lysates were hybridized with two probes, a 32P-labeled RNA "label probe" which provided signal and an oligodeoxyribonucleotide "capture probe" containing a poly(dA) tail which provided a mechanism for selective purification. Ternary hybrids were "captured" on oligo(dT)-coated superparamagnetic beads through a readily reversible interaction with the poly(dA) of the capture probe. RNA did not bind to dT beads through poly(A) under the capture conditions used. Hybrids were purified through cycles of capture on and release from dT beads, with each cycle yielding a 100- to 1000-fold reduction in noise (unhybridized label probe) and a 50-90% recovery of signal (hybridized label probe). Noise was driven below detectable limits after three cycles of capture, thereby improving the sensitivity of measuring target RNA. As few as 15,000 target molecules, 15 fg of a 3-kb RNA, was detectable in the equivalent of 2 x 10(6) cells in concentrated cell lysates (10(8) cells/ml). Since hybridization with both probes was required in order to yield a signal, hybridization specificity could be adjusted with either or both probes. The greater specificity and lack of noise increased confidence that the signal was proportional to the amount of RNA of interest.     

Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot

The northern blot, or RNA gel blot, is a widely used method for the discovery, validation and expression analysis of small regulatory RNA such as small interfering RNA (siRNA), microRNA (miRNA) and piwi-interacting RNA (piRNA). Although it is straightforward and quantitative, the main disadvantage of a northern blot is that it detects such RNA less sensitively than most other approaches. We found that the standard dose of UV used in northern blots was not the most efficient at immobilizing small RNA of 20–40nt on nylon membranes. However, increasing the dose of UV reduced the detection of miRNA by hybridization in northern blotting experiments. We discovered that using the soluble carbodiimide, EDC, to cross-link RNA to nylon membranes greatly improved the detection of small RNA by hybridization. Compared to standard UV cross-linking procedures, EDC cross-linking provided a 25–50-fold increase in the sensitivity of detection of siRNA from plants and miRNA or piRNA from mammalian cells. All types of hybridization probes tested benefited from the new cross-linking procedure. Cross-linking was dependent on a terminal phosphate and so, should be applicable to other related categories of small RNA.     

Follicle-stimulating hormone regulation of cytochrome P-450 side-chain cleavage messenger ribonucleic acid accumulation by porcine granulosa cells isolated from small and medium follicles

The experiments described here were conducted to examine regulation of cytochrome P-450 side-chain cleavage (SCC) mRNA accumulation in porcine granulosa cells isolated from small (1-4-mm) and medium (5-6-mm) follicles. Granulosa cells were cultured under the following conditions: 1) for 48 h or 96 h with 0, 50, or 200 ng/ml porcine FSH; 2) for 96 h with 200 ng/ml FSH and aminoglutethimide (100 microM); and 3) for 96 h with forskolin (100 microM). Total RNA was extracted and examined by Northern and dot-blot hybridization analysis, and culture media were assayed for progesterone concentration. Northern blot analysis revealed a single band approximately 2.1 kb in size. Accumulation of SCC mRNA by granulosa cells was both FSH dose- and culture time-dependent (p less than 0.05) with maximal increases approximately 4.5 times control levels. Aminoglutethimide reduced progesterone production by about 80% while having no effect on granulosa cell accumulation of SCC mRNA compared to cells stimulated with 200 ng/ml of FSH. Forskolin-treated cells produced significantly more progesterone than did cells treated with FSH, but accumulation of SCC mRNA was similar. In response to FSH, concentration of SCC mRNA did not vary with follicle size, but granulosa cells from small follicles produced significantly more progesterone than did those from medium follicles. These results demonstrate that concentration of SCC mRNA in cultured porcine granulosa cells is FSH dose-dependent, does not vary significantly in cells from small- and medium-sized follicles, and is correlated with progesterone production, but may not parallel progesterone secretion. This last observation indicates that control at sites other than SCC mRNA can affect progesterone production.     

Rapid and quantitative preparation of cytoplasmic RNA from small numbers of cells

An extremely simple procedure for preparing cytoplasmic RNA from small numbers of cells is described. Cells are lysed with the detergent NP-40 and efficient extraction of protein from the postnuclear cytoplasmic lysate is ensured by denaturation with sodium dodecyl sulfate and urea. This procedure is suitable for preparing RNA from many cell types. All procedures have been scaled down to be performed in 1.5-ml microfuge tubes and thus RNA may be prepared from small numbers of cells. The procedure is extremely rapid and RNA is ready for Northern gel analysis in less than 30 min. Because so few steps are involved, RNA recovery is quantitative.     

Use of cervical Cytobrush samples for dot-blot detection and Southern blot typing of human papillomaviruses using subgenomic probes

The use of Cytobrush samples for the detection and typing of cervical human papillomavirus (HPV) infections was analyzed. The average yield of squamous cells from Cytobrush samples was on the order of 10(6) cells when the sample was collected in a lytic collection buffer, which was approximately double the content of (1) samples collected in ethanol and (2) an average biopsy specimen. The material obtained could be used for sensitive detection and typing of HPV infections using, respectively, a nonradioactive dot-blot method and the Southern blot procedure performed with subgenomic probes, which permitted a simple interpretation even in cases of mixed infections. A sample containing at least 500,000 viral copies was required for the detection and typing. At this level of sensitivity, the frequencies of HPV obtained in different risk groups varied from 6% (in "healthy" young women) to 53% (in women with abnormal cytologic findings in simultaneous smears). The noninvasive nature of the sampling procedure and the relative simplicity of the test should allow this method to be applied in large-scale studies.