Confidence intervals for a variance ratio, or for heritability, in an unbalanced mixed linear model

A procedure is presented for constructing an exact confidence interval for the ratio of the two variance components in a possibly unbalanced mixed linear model that contains a single set of m random effects. This procedure can be used in animal and plant breeding problems to obtain an exact confidence interval for a heritability. The confidence interval can be defined in terms of the output of a least squares analysis. It can be computed by a graphical or iterative technique requiring the diagonalization of an m X m matrix or, alternatively, the inversion of a number of m X m matrices. Confidence intervals that are approximate can be obtained with much less computational burden, using either of two approaches. The various confidence interval procedures can be extended to some problems in which the mixed linear model contains more than one set of random effects. Corresponding to each interval procedure is a significance test and one or more estimators.     

Comparison of Two Procedures for Detection of Salmonella in Food, Feed, and Pharmaceutical Products

A survey of food, feed, and pharmaceutical products was undertaken to compare an accelerated Salmonella detection procedure by enrichment serology (ES) to the traditional procedure outlined in the Bacteriological Analytical Manual (BAM). Excellent agreement between the two methods was obtained in the results of 689 test samples involving 35 different products. In addition to being more rapid and simpler to perform, the ES procedure was just as accurate and sensitive as the BAM procedure.     

A berberine-aniline blue fluorescent staining procedure for suberin,lignin, and callose in plant tissue

Summary A fluorescent staining procedure to detect suberin, lignin and callose in plants has been developed. This procedure greatly improves on previous methods for visualizing Casparian bands in root exodermal and endodermal cells, and performs equally well on a variety of other plant tissues. Berberine was selected as the most suitable replacement forChelidonium majus root extract after comparing the staining properties of the extract with those of four of its constituent alkaloids. Aniline blue counterstaining efficiently quenched unwanted background fluorescence and nonspecific berberine staining, while providing a fluorochrome for callose. When used with multichambered holders which allow simultaneous processing of freehand sections, this efficient staining procedure facilitates morphological studies involving large numbers of samples.Abbreviations ISCC-NBS Inter-Society Color Council-National Bureau of Standards - UV ultraviolet light     

Magnetic, microplate-format plasmid isolation protocol for high-yield, sequencing-grade DNA

We have developed a rapid, microplate-format plasmid isolation procedure to purify sequencing-grade DNA templates for high-throughput DNA sequencing operations. A modified lysozyme/boiling method is used to produce a plasmid-containing supernatant that is then purified by iron bead capture. After binding, the beads are pelleted in a magnetic field, washed and the DNA eluted in water. The method yields up to 10 micrograms plasmid DNA from a 1-mL overnight culture in a deep-well microplate. The procedure is suitable for large-scale experiments, amenable to automation and does not require expensive reagents or equipment. The entire protocol can be completed in as little as 2 h, and one technician with a 96-well pipetting station can process up to 48 plates per day. This protocol is ideal for any high-throughput operation in which template quantity, quality and reproducibility are of primary importance.     

Evaluation of a Fluorescent Antibody-Enrichment Serology Combination Procedure for the Detection of Salmonellae in Condiments, Food Products, Food By-Products, and Animal Feeds

The reliability of the enrichment serology (ES), fluorescent antibody (FA), and a combination of the FA and ES procedures for the detection of salmonellae were compared to the Salmonella cultural procedure outlined in the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM). A total of 126 subsamples from 22 different products were analyzed. By utilizing the BAM procedure as the reference standard, a total of 66 samples were positive for salmonellae. Within 44 h approximately 65% of the Salmonella-negative samples could be cleared by the FA test. At the end of 50 h 97% of the Salmonella-negative samples could be cleared by the combination FA-ES test. The FA procedure detected all 66 BAM positives but exhibited a high incidence of presumptive positives which were cultural negatives. The ES procedure detected 64 of the 66 BAM positives but exhibited a low incidence of presumptive positives which were cultural negatives. Incorporating positive FA and positive ES results in a combination FA-ES technique revealed that FA-ES positives were statistically equivalent to BAM positives.     

Precise, easy measurement of glass pipet tips for microinjection or electrophysiology

We describe a simple procedure to measure precisely the tip diameter of glass micropipets. This procedure can improve the precision of microinjection and some electrophysiological measurements due to their extreme sensitivity to the pipet's exact geometric dimensions. The technique is nondestructive and readily carried out as a brief, extra step in these applications. Digital measurements of the threshold pressures for gas bubbling from (gas filled) pipets submerged in methanol were used to calculate the corresponding inner tip diameters using the LaPlace equation. Direct measurements of the inner tip diameters (from 0.2-5.0 mu) using scanning electron microscopy confirmed the validity of this indirect, simple procedure.     

Adapters, shims, and glue--service interoperability for in silico experiments

MOTIVATION: Computationally, in silico experiments in biology are workflows describing the collaboration of people, data and methods. The Grid and Web services are proposed to be the next generation infrastructure supporting the deployment of bioinformatics workflows. But the growing number of autonomous and heterogeneous services pose challenges to the used middleware w.r.t. composition, i.e. discovery and interoperability of services required within in silico experiments. In the IRIS project, we handle the problem of service interoperability by a semi-automatic procedure for identifying and placing customizable adapters into workflows built by service composition. RESULTS: We show the effectiveness and robustness of the software-aided composition procedure by a case study in the field of life science. In this study we combine different database services with different analysis services with the objective of discovering required adapters. Our experiments show that we can identify relevant adapters with high precision and recall.     

A simple, versatile, nondisruptive method for the isolation of morphologically and chemicaly pure basement membranes from several tissues.

A simple procedure has been developed for the isolation of ultrastructurally pure, intact basement membranes from bovine retinal and brain blood vessels, rabbit renal tubules and rat renal glomeruli. By this procedure, cell membranes and intracellular materials are selectively solubilized with 4% sodium deoxycholate to yield morphologically and chemically intact basement membrane preparations. Therefore, this method appears to be a versatile, nondisruptive procedure for the isolation and characterization of basement membranes from a variety of tissues. Its applicability has been demonstrated by the preparation for the first time of isolated basement membranes from non-renal mammalian blood vessels.     

A simple, rapid and quantitative method for preparing Arabidopsis protein extracts for immunoblot analysis

Although Arabidopsis has numerous well documented advantages for genetic and molecular analyses, its small size can be a limitation for biochemical and immunochemical assays requiring protein extraction. We have developed a rapid method to extract total protein from small amounts of Arabidopsis tissue that can be used for quantitative immunoblot analysis. The procedure involves direct extraction of tissue into SDS-containing buffer under conditions permitting immediate protein quantification in the extract, using commercially available kits without prior fractionation. This approach provides maximal extraction and quantitative recovery of total cellular protein, together with accurate evaluation of target protein levels as a proportion of the total. We have examined the utility and sensitivity of the procedure using monoclonal antibodies to phytochromes A and C (phyA and phyC), which are high- and low-abundance members, respectively, of the phytochrome family in Arabidopsis. Both phytochromes could be rapidly and readily quantified in the tissues examined, with phyC being detectable in extracts representing as few as five dark-grown seedlings, two light-grown seedlings, or half a single leaf from 3-week-old adult plants. The data indicate that the procedure may have broad utility for the detection and quantitative analysis of many proteins, including those of low abundance, in a variety of applications in Arabidopsis. In one such application, we used transgenic Arabidopsis phyC-overexpressor seedlings to demonstrate that the procedure can be used to detect transgene-encoded protein early at the segregating T2 generation, thereby offering the capacity for accelerated screening and selection of lines engineered to overexpress target proteins.     

Native Species for Roadslope Revegetation: Selection,Validation, and Cost Effectiveness

Native species arise as an alternative to improve the poor success of traditional hydroseeding with commercial species in degraded areas. The objectives of this study were to (1) establish a procedure to select suitable native species for roadslope revegetation, (2) validate the procedure by means of field sowing experiments, and (3) assess cost effectiveness of the use of native species in hydroseeding as regards commercial ones. Vegetation surveys were performed in semiarid roadslopes of East Spain and species success evaluated according to abundance and frequency. A list of potentially suitable species for revegetation was produced and a subset of species selected and hydroseeded to check their suitability in hydroseeding. Cost effectiveness of the use of native species was estimated. Results support the suitability of the procedure for the selection of native species. Vegetation cover produced by the hydroseeded mixture of native species was high (between 43 and 70% throughout the 4 years) and was 4–20 times higher than the cover produced by a standard commercial seed mixture. The price of the selected seed mixture was 30 times that of the commercial one, but the cost of relevant ecological advantages provided by the use of native species was only twice that of the commercial species on the basis of cost effectiveness assessment. Practical consequences for restorers, policymakers, and practitioners are discussed, and the use of native species is encouraged to improve revegetation success of roadslopes and similar slope types especially in semiarid and arid environments.     

A rapid procedure for the isolation, identification and estimation of citrinin

A rapid procedure for the isolation, identification, and estimation of citrinin, the antibiotic cum nephrotoxin, has been proposed. The colorimetric procedure proposed for estimation makes use of the phenolic function of the compound. This method was found to be applicable to a wide variety of materials.     

A simplified and efficient method for in situ hybridization to whole Drosophila embryos, using electrophoresis for removing non-hybridized probes

We report a simplified and reliable method for non-radioactive in situ hybridization to whole Drosophila embryos. In the previous method (Tautz and Pfeifle, 1989) the post-hybridization wash, or the procedure for washing non-hybridized probe away from embryos depends simply on diffusion. We modified the method with application of electrophoresis to the wash. After hybridized with RNA probe, embryos were transferred to a small well where an electric charge was given to drive non-hybridized probe away from the embryos. This procedure enables us to acquire a much higher signal-to-noise ratio than that obtained from a conventional method. Furthermore, this is a time-saving method. We propose a term "electro-wash" for this procedure.     

A simple, sensitive radiomicroassay for 5'-nucleotidase

The reagent 9,10-phenanthrenequinone has been shown to react with free arginine or with arginine residues within proteins to produce a compound whose fluorescence can be used to quantitatively determine submicrogram amounts of arginine. The assay procedure, which is simple, convenient, and suitable for automation, is performed by mixing a slight excess of phenanthrenequinone with the sample at high pH followed by acidification to produce the fluorescence. None of the commonly occurring amino acids were found to interfere with the analysis. Several commonly used buffers and organic solvents also did not interfere. The arginine content of intact proteins was accurately determined by this procedure with only microgram quantities of protein required. The method is compared with other commonly used procedures for arginine and protein determination.     

Novel isolation procedure for short-, medium-, and long-chain acyl-coenzyme A esters from tissue

A novel procedure for the quantitative isolation and purification of acyl-coenzyme A esters is presented. The procedure involves two steps: (1) tissue extraction using acetonitrile/2-propanol (3+1, v+v) followed by 0.1 M potassium phosphate, pH 6.7, and (2) purification using 2-(2-pyridyl)ethyl-functionalized silica gel. Recoveries determined by adding radiolabeled acetyl-, malonyl-, octanoyl-, oleoyl-, palmitoyl-, or arachidonyl-coenzyme A to powdered rat liver varied 93-104% for tissue extraction and 83-90% for solid-phase extraction. The procedure described allows for isolation and purification, with high recoveries, of acyl-coenzyme A esters differing widely in chain length and saturation.     

A sensitive, precise, and convenient method for determination of 1,25-dihydroxyvitamin D in human plasma.

A new, highly sensitive and relatively convenient method has been developed for the determination of 1,25-dihydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₂ in blood plasma. The method involves a simplified and more specific extraction procedure, new rapid and effective methods of purification, and a competitive binding assay using intestinal cytosol from rachitic chicks. The method also includes a procedure for stabilizing the cytosol binding protein and a convenient procedure for the separation of bound from free 1,25-dihydroxyvitamin D₃ with the use of polyethylene glycol. The recovery of 1,25-dihydroxyvitamin D₃ during extraction and purification is 68% and triplicate determinations can be made on a 5-ml plasma sample. With this method, rachitic chick plasma, plasma from anephric patients, and plasma from patients suffering severe endstage renal failure show no detectable 1,25-dihydroxyvitamin D, while normal human values have been found to be 29 ± 2 pg/ml.     

A robust, streamlined, and reproducible method for proteomic analysis of serum by delipidation, albumin and IgG depletion, and two-dimensional gel electrophoresis

Serum is a readily available source for diagnostic assays, but the identification of disease-specific serum biomarkers has been impeded by the dominance of human serum albumin and immunoglobulins (Igs) in the serum proteome. There is a need to reduce the technical variation in serum processing and analysis to allow for a reproducible analysis of large cohorts. To this end, we have developed a rapid and reproducible procedure for sample preparation and high-resolution two-dimensional gel electrophoresis to analyze human serum. Serum is centrifuged at high speed to remove lipids and aggregated proteins, incubated with protein G resin to remove IgG, precipitated with NaCl/ethanol to deplete albumin, and slowly resolubilized in a sodium dodecyl sulfate (SDS)/N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES) buffer. The delipidated and IgG/albumin depleted serum proteins are focused on pH 4-7 linear large immobilized pH gradient strips, and then resolved by Bis-Tris SDS-polyacrylamide gel electrophoresis. The robustness and reproducibility of the optimized procedure was determined for three individual serum samples on three consecutive days. An image analysis of the nine silver-stained gels demonstrated that the intensity and localization of protein spots are highly reproducible. Our IgG and albumin depletion procedure will aid in screening the patient sera for normal biological variation and disease-specific biomarkers.     

A rapid and accurate procedure for assaying DNA polymerase,RNA polymerase,or ribosome dependent protein synthesis

A descending paper chromatographic procedure for separating trichloracetic acid soluble from precipitable material is described. Its use for assaying DNA, RNA, or protein synthesis from a variety of sources with various states of purity attests to its general applicability to reactions of interest to molecular biologists. Large numbers of analyses can be carried out in a short period of time without compromising accuracy or reliability. The use of this procedure for still more enzymes, those involved in modifying DNA, RNA, or proteins, is discussed.     

A modified procedure for quantitative analysis of mtDNA, detecting mtDNA depletion

Quantitative analysis of mitochondrial DNA (mtDNA) is crucial for proper diagnosis of diseases that are caused by or associated with mtDNA depletion. However, such a quantitative characterization of mtDNA is not a simple procedure and requires several laboratory steps at which potential errors can accumulate. Here, we describe a modified procedure for quantitative human mtDNA analysis. The procedure is based on using two PCR-amplified, fluorescein-labeled DNA probes, complementary to mtDNA (detection probe) and chromosomal 18S rDNA (reference probe), both of similar length. Thus, equal amounts of these probes can be used and, contrary to previously published procedures, no mtDNA purification (apart from total DNA isolation) or 18S rDNA cloning is necessary for probe preparation. Two separate hybridizations (each with one probe) are suggested instead of one hybridization with both probes; this decreases background signals and enables adjustment of the strength of specific signals from both probes, which is useful in the subsequent densitometric analysis after superimposing of both pictures. Using different DNA amounts for reactions, we have proved that the procedure is quantitative in a broad range of sample DNA concentrations. Moreover, we were able to detect mtDNA depletion unambiguously in tissue samples from patients suffering from diseases caused by dysfunction of mtDNA.     

A filtration, incubation and staining reactor including a new protocol for FISH

A newly developed device for performing fluorescence in situ hybridization (FISH) is described. An adapted procedure was compared with two typical FISH protocols. Tests were performed with Pseudomonas cells and the gene probe EUB338. With the novel procedure, we obtained a better recovery of cells and less variability in results.