A practical approach for quantitating specific mRNAs by solution hybridization

The preparation and use of a specific cDNA probe for quantitating mRNA by solution hybridization is described. Cloned DNA sequences are nick translated, denatured, hybridized to single-stranded M13 clones containing message strand (mDNA) sequences, and separated chromatographically on Bio-Gel A50 under first native and then denaturing conditions to yield a single-stranded cDNA probe. The details of a solution hybridization assay in which the single-stranded cDNA is used to quantitate mRNA in total nucleic acid samples are described. As little as 0.5 pg of mRNA can easily be detected within a day of sample isolation. Thus, the assay is both rapid and sensitive and can be used to measure RNAs complementary to any cloned DNA sequence. It is ideally suited to situations when accurate quantitation of multiple samples is anticipated.     

A quick method of determining rock surface area for quantification of the invertebrate community

Stone and rock substrates provide important habitat for many types of stream-dwelling invertebrates. Measures of the invertebrate communities inhabiting rock substrates are often an important component of ecological, monitoring and disturbance studies in streams. A major obstacle to researchers examining rock-inhabiting invertebrates is the time and effort expended on currently used methods of determining rock surface area to derive invertebrate densities on these substrates. In an attempt to more efficiently determine invertebrate densities from rock substrates in streams, we tested a direct method of calculating rock surface area from rock weight or displacement volume. This method allows very quick determinations of rock surface area in the field. Surface area estimates made using this technique were highly correlated to those from a widely used and more time-consuming method. Measurements made using this new method should theoretically give better surface area estimates than any other commonly used technique.     

A novel method for determining linkage between DNA sequences: hybridization to paired probe arrays

Cooperative hybridization has been used to establish physical linkage between two loci on a DNA strand. Linkage was detected by hybridization to a new type of high-density oligonucleotide array. Each synthesis location on the array contains a mixture of two different probe sequences. Each of the two probes can hybridize independently to a different target sequence, but if the two target sequences are physically linked there is a cooperative increase in hybridization yield. The ability to create and control non-linear effects raises a host of possibilities for applications of oligonucleotide array hybridization. The method has been used to assign linkage in 50:50 mixtures of DNA containing single nucleotide polymorphisms (SNPs) separated by 17, 693, 1350 and 2038 bp and to reconstruct haplotypes. Other potential uses include increasing the specificity of hybridization in mutation detection and gene expression monitoring applications, determining SNP haplotypes, characterizing repetitive sequences, such as short tandem repeats, and aiding contig assembly in sequen-cing by hybridization.     

A method for determining in peacultures,the amount of molecular nitrogen fixed and the amount of combined nitrogen taken up from the soil

Summary A method has been described for the determination of the rate of nitrogen fixation when peas are grown in a soil from which they can take up combined nitrogen. This method is based on an earlier observation that previous nodule formation by an ineffective strain of the peaRhizobium H VIII prevents later nodulation by effective strains. Parallel pot experiments (muddy clay soil) in which pea plants were inoculated either with the ineffective strain VIII or with the effective strain H 47, showed that no effective nodules were produced by the former within a period of 2 months. Differences between the nitrogen contents of the peas in the two cultures can therefore be assumed to indicate the amount of atmospheric nitrogen fixed. Two successive oat crops were grown after the peas in order to determine the after-effect of the peas.The oats took up from the soil much more nitrogen than did the peas.     

Use of exhaustive nucleic acid hybridization for determining the amount and size distribution of newly synthesized bacterial message RNA

An RNA-DNA hybridization method is described for determining the fraction of a radioisotopically labeled RNA preparation isolated from bacteria that is message RNA. Experiments are performed under conditions where over 95% of the input RNA is converted to an RNA-DNA complex. Competitive hybridization methods are described that partition the RNA-DNA hybrids into ribosomal RNA-DNA and nonribosomal RNA-DNA hybrids. Evidence is presented that the competition is specific. Message RNA radioactivity was assumed to be RNA radioactivity not competed by ribosomal RNA and transfer RNA. The fraction of RNA made at a given instant under balanced growth conditions (30 C, in minimal medium), that is, message RNA, was determined to be 65%. The method was also used to measure the size distribution of newly synthesized mRNA in Escherichia coli. That size was found to average 3–8 × 10⁵ daltons.This research was supported by grant GM-14368 from the Institute of General Medical Sciences, National Institutes of Health. Two of the authors (D. P. and A. J.) were supported by predoctoral fellowships from the National Institute of General Medical Sciences.     

Fluorescence in situ hybridization method for co-localization of mRNA and GEP

Co-localization studies using green fluorescent protein (GFP) and fluorescence immunohistochemistry have become commonplace. However, co-localization studies using GFP and mRNA in situ hybridization are rare, in large part because typical in situ hybridization reaction conditions often lead to the loss of GFP fluorescence. Here, we describe a new fluorescence mRNA in situ hybridization protocol using cRNA riboprobes that leaves GFP fluorescence intact. This protocol is based on a urea-based hybridization buffer and the Tyramide Signal Amplification system. This protocol should provide researchers engaged in the use of GFP with a solid starting point for adapting their own in situ hybridization protocols.     

Rapid method for determining the stability of streptomycin sulfate powders]

A method applicable for beforehand determination of streptomycin sulfate powder stability by the colour of its solutions is described. It is shown that the results of such a determination satisfactorily coincided with the results of indirect determination of the solution colour after prolonged storage of the powders at room temperature.     

Improved method for cloning DNA complementary to minor mRNAs: preparation of a hybridization probe from purified mRNAs encoding intermediate filament proteins

A procedure for the rapid fractionation of mRNA has been used to enrich mRNAs encoding a set of intermediate filament proteins in trophoblastoma cells. The procedure involves sucrose-gradient fractionation followed by high-resolution preparative gel electrophoresis. Part of the enriched mRNA preparation has been used to prepare a hybridization probe to screen a trophoblastoma cDNA library in Escherichia coli. A small proportion of the clones hybridized to the probe, and among these a specific clone was identified.     

Correlation between intrinsic mRNA stability and the affinity of AUF1 (huRNP D) and HuR for A+U-rich mRNAs

Presence of A+U-rich elements (AREs) within 3-untranslated regions (3UTRs) of numerous mRNAs has been associated with rapid mRNA turnover; however, the interaction of specific factors with AREs is also associated with mRNA stabilization. Recently, two ARE binding proteins with putative mRNA destabilizing (AUF1) and stabilizing (HuR) properties have been described. However, no direct comparison of AUF1 and HuR binding properties has been made. Therefore, we examined the relative affinities of p37AUF1 and HuR for a diverse set of ARE-containing mRNAs encoding -adrenergic receptors, a proto-oncogene, and a cytokine. We find that high-affinity AUF1 binding appears to require elements beyond primary nucleotide sequence. In contrast, binding of HuR appears considerably less constrained. As a functional correlate, we determined the ability of these specific mRNA sequences to affect the stability of chimeric -globin mRNA constructs. Although the relative affinity of AUF1 and HuR are generally predictive of mRNA stability, we find that certain mRNA sequences do not conform to these generalizations.     

A method for bacteriocin quantification

Different aspects of the most commonly used assay methods in the study of bacteriocins were examined. The conditions under which extraction and incubation (including exposure time) take place were analysed, and several different formal models that are usually employed to calculate ID50 were compared. As an alternative designed to overcome the problems which characterize the response of micro-organisms that are sensitive to bacteriocins, an operative procedure in a liquid medium and a modified re-parameterized logistic equation is proposed. When applied to the inhibition of Leuconostoc mesenteroides by nisin, the model allows an optimal experimental procedure to be defined.     

Fluorescence in situ hybridization: a new method for determining primary sex ratio in ants

The haplodiploid sex determining system in Hymenoptera, whereby males develop from haploid eggs and females from diploid eggs, allows females to control the primary sex ratio (the proportion of each sex at oviposition) in response to ecological and/or genetic conditions. Surprisingly, primary sex ratio adjustment by queens in eusocial Hymenoptera has been poorly studied, because of difficulties in sexing the eggs laid. Here, we show that fluorescence in situ hybridization (FISH) can be used to accurately determine the sex (haploid or diploid) of eggs, and hence the primary sex ratio, in ants. We first isolated the homologue coding sequences of the abdominal-A gene from 10 species of 8 subfamilies of Formicidae. Our data show that the nucleotide sequence of this gene is highly conserved among the different subfamilies. Second, we used a sequence of 4.5 kbp from this gene as a DNA probe for primary sex ratio determination by FISH. Our results show that this DNA probe hybridizes successfully with its complementary DNA sequence in all ant species tested, and allows reliable determination of the sex of eggs. Our proposed method should greatly facilitate empirical tests of primary sex ratio in ants.     

Prolonged hybridization with a cRNA probe improves the signal to noise ratio for in-tube in situ hybridization for quantification of mRNA after fluorescence-activated cell sorting

We developed an in-tube in situ hybridization method for mRNA quantification after fluorescence-activated cell sorting (FACS-mQ). A specific RNA in a particular cell type is stained with a cRNA probe and a fluorescent dye, which allows the stained cells to be selected by FACS without excessive RNA degradation. Our previous protocol required 4 h for hybridization with a cRNA probe, which might not produce enough fluorescence signal for sorting genes with low expressions. We determined the effect of prolonged hybridization for in-tube in situ hybridization on quantitative measurement of intracellular RNAs. During the hybridization step, the quantity of ACTB mRNA decreased gradually until 4 h, but remained constant from 4 to 16 h below 63.6° C. For flow cytometry, cells hybridization with cRNA probes for TG mRNA at 60° C for 16 h showed both increased signal and decreased background fluorescence compared to those hybridized for 4 h. These results indicate that when performing in-tube in situ hybridization, hybridization temperature can be raised to 63.6° C and the hybridization step can be extended up to 16 h without excessive intracellular RNA degradation.     

DNA multiplex hybridization on microarrays and thermodynamic stability in solution: a direct comparison

Hybridization intensities of 30 distinct short duplex DNAs measured on spotted microarrays, were directly compared with thermodynamic stabilities measured in solution. DNA sequences were designed to promote formation of perfect match, or hybrid duplexes containing tandem mismatches. Thermodynamic parameters ΔH°, ΔS° and ΔG° of melting transitions in solution were evaluated directly using differential scanning calorimetry. Quantitative comparison with results from 63 multiplex microarray hybridization experiments provided a linear relationship for perfect match and most mismatch duplexes. Examination of outliers suggests that both duplex length and relative position of tandem mismatches could be important factors contributing to observed deviations from linearity. A detailed comparison of measured thermodynamic parameters with those calculated using the nearest-neighbor model was performed. Analysis revealed the nearest-neighbor model generally predicts mismatch duplexes to be less stable than experimentally observed. Results also show the relative stability of a tandem mismatch is highly dependent on the identity of the flanking Watson–Crick (w/c) base pairs. Thus, specifying the stability contribution of a tandem mismatch requires consideration of the sequence identity of at least four base pair units (tandem mismatch and flanking w/c base pairs). These observations underscore the need for rigorous evaluation of thermodynamic parameters describing tandem mismatch stability.     

Quantitation of GLUT1 and GLUT4 mRNA using a solution hybridization assay

The development of a solution hybridization assay for detecting GLUT1 and GLUT4 mRNA is described. The details of this assay are described in which copy RNA is used to quantitate messenger RNA in total RNA samples. This solution hybridization assay is highly specific and reproducible and is significantly more sensitive than Northern blotting. Since GLUT mRNAs can be quantitated in as little as 25 mg tissue, this technique is essential when the supply of tissue is limited. Furthermore, the elimination of gel-based separation techniques allows for mRNA quantitation in several hundred samples within two days following isolation of samples.     

16S rRNA targeted sandwich hybridization method for direct quantification of mycobacteria in soils

Boreal soils have been suspected reservoirs of infectious environmental mycobacteria. Detection of these bacteria in the environment is hampered by their slow growth. We applied a quantitative sandwich hybridization approach for direct detection of mycobacterial 16S rRNA in soil without a nucleic acid amplification step. The numbers of mycobacterial 16S rRNA molecules found in the soil indicated the presence of up to 10(7) to 10(8) mycobacterial cells per gram of soil. These numbers exceed by factor of 10 to 100 x the previous estimates of mycobacteria in soil based on culture methods. When real-time PCR with mycobacteria targeting primers was used to estimate the number of 16S rDNA copies in soil, one copy of 16S rDNA was detected per 10(4) copies of 16S rRNA. This is close to the number of 16S rRNA molecules detected per cell by the same method in laboratory pure cultures of M. chlorophenolicum. Therefore a major part of the mycobacterial DNA in the studied soils may thus have represented metabolically active cells. The 16S rRNA sandwich hybridization method described in this paper offers a culture independent solution for tracking environmental reservoirs of viable and potentially infectious mycobacteria.