A method for preparing freeze-clamped tissue samples for metabolite analyses

A rapid and simple method for preparing freeze-clamped tissue samples for metabolite determinations is described. Freeze-clamped rat heart tissue samples weighing from 0.8 to 1.0 g were homogenized directly in an Ultra-Turrax homogenizer for 60 s in 3.5 ml of ice-cold 0.6 M HClO4 without pulverizing them in liquid nitrogen. After centrifugation, the pellet was rehomogenized in the Ultra-Turrax homogenizer for 30 s in 1.5 ml of HClO4. Following a further centrifugation the extracts were combined and the pH was adjusted to 7.0 by adding 5 M K2CO3. The neutralized supernatant was used for the desired assays. The analyses of the tissue extracts obtained from isolated perfused rat hearts by the present method give similar results for different kinds of metabolites than those processed according to the previous classical method. Moreover, the values of the various parameters determined from the tissue extracts prepared according to the method described here are similar to the data reported in literature. The method can be readily applied to any other freeze-clamped tissue. The greatest improvement obtained is that the homogenization procedure can be accomplished easily and conveniently in about one-tenth of the time required for the earlier classical method without the time-consuming and unpleasant tissue grinding in liquid nitrogen.     

A Method for the Preparation of Platelets for Transmission Electron Microscopy

A modified method for the preparation of platelets for transmission electron microscopy has been developed. A suspension of platelets in plasma is fixed in glutaraldehyde, immobilized in agarose, and further fixed in osmium tetroxide. The specimen is then dehydrated with alcohol and embedded in Spurr. The key point of this method is the immobilization of the platelet pellet in agarose gel, thus dispensing with the difficulties associated with excessive centrifugation and resuspension of the platelets. Platelets prepared for transmission electron microscopy by this method show excellent preservation of ultrastructure. In addition, this method is relatively rapid, requiring only one day for processing the specimen.     

A simple method for classifying genes and a bootstrap test for classifications

A new simple method for classifying genes is proposed based on Klastorin's method. This method classifies genes into monophyletic groups which are made distinct from each other by evolutionary changes. The method is applicable as long as the phylogenetic tree of genes is obtained. There is a fast algorithm for obtaining the classification. A bootstrap test of a classification is also presented. As an example, we classified opsin genes. The classification obtained by this method is the same as the previous classification based on the function of opsins.     

A new method for predicting the opening angle for soft tissues

The purpose of this paper is to present a simple new method for calculating the opening angle produced by a given residual stress field in a soft biological tissue. The method uses minimization of potential energy, and is therefore named the MPE method. The accuracy of the MPE method is evaluated by comparing the opening angle it predicts to results from a finite element model of the opening angle experiment. We show that the MPE method provides good predictions of the opening angle, and that it is significantly more accurate than two other methods previously used in the literature.     

A procedure for analysis of stopped-flow transients for protein-ligand association

A method for extracting kinetic and optical parameters from progress curves for protein-ligand association, obtained by stopped-flow experiments, is described. The method is limited to one-step and two-step association kinetics, but it allows concentration of protein and offset of the signals to be adjustable parameters during an interactive non-linear least-squares fitting procedure. The method is tested on simulated pseudo-experimental data and applied to progress curves obtained in a stopped-flow spectrofluorimeter, for association of the translation initiation factor eIF4E with 7-methyl-GDP, an analog of 5'-end of mRNA.     

A new graphic method for determining the rate constant for affinity of a ligand for its receptor]

A new method of determination of the equilibrium constant for a ligand binding to acceptor and evaluation of the number of binding sites on the acceptor molecules (or cells) is suggested. The method is simpler, more convenient, and more precise than Klotz's or Scatchard's method.     

Evaluation of a cushioned method for centrifugation and processing for freezing boar semen

The purpose of this investigation was to evaluate the use of an iodixanol cushion during centrifugation on sperm recovery and yield after centrifugation (sperm recovery, sperm motility, viability, membrane lipid disorder, acrosome reaction and ROS generation); and to investigate how this procedure affects sperm function after freezing-thawing (sperm motility, membrane lipid disorder, acrosomal status and homologous in vitro penetration test). The sperm-rich fractions from fertile boars were centrifuged under two centrifugation régimes: 800xg for 10min (standard method) and 1000xg for 20min with an iodixanol (60% w/v) cushion at the bottom of the centrifuge tubes (Cushion method). The highest recovery was achieved using the cushion method (sperm loss for cushion method was 0.50%+/-0.18 versus 2.97%+/-0.43 for standard method, P<0.01) and sperm quality was not significantly affected by the centrifugation régime. The motion parameters (% progressive motility, % motility, VCL, VSL, VAP, ALH, BCF, P<0.05) of frozen-thawed samples showed higher values using the standard method. However, a higher number of viable spermatozoa with lower lipid disorders were found in spermatozoa processed with the cushion method. The in vitro penetration assay showed that the individual boar influenced the parameters studied but there were no differences between the two centrifugation régimes used. Our results support the hypothesis that the proportion of sperm loss in frozen-thawed semen was significantly influenced by the centrifugation régime. Therefore, the iodixanol cushion method is a suitable tool for cryopreservation of boar semen in order to reduce sperm loss without affecting sperm quality.     

Curtis' Substitute for Van Gieson Stain

A triple staining method is described in which nuclear staining is by Weigert's hematoxylin. The cytoplasmic and collagen staining is effected by the Curtis substitute for Van Gieson, in which ponceau S is substituted for acid fuchsin. Nuclear staining is sharper than with Delafield's hematoxylin. The red of the collagen fibers is probably not subject to fading. Unlike Van Gieson, this method gives staining of reticular as well as collagen fibers. The advantages of the method are its simplicity and reliability. The use of this method is made possible by a new source of reliable samples of the ponceau S called for in this method.     

Optimization for peptide sample preparation for urine peptidomics

Analysis of native or endogenous peptides in biofluids can provide valuable insights into disease mechanisms. Furthermore, the detected peptides may also have utility as potential biomarkers for non-invasive monitoring of human diseases. The non-invasive nature of urine collection and the abundance of peptides in the urine makes analysis by high-throughput ‘peptidomics’ methods , an attractive approach for investigating the pathogenesis of renal disease. However, urine peptidomics methodologies can be problematic with regards to difficulties associated with sample preparation. The urine matrix can provide significant background interference in making the analytical measurements that it hampers both the identification of peptides and the depth of the peptidomics read when utilizing LC-MS based peptidome analysis. We report on a novel adaptation of the standard solid phase extraction (SPE) method to a modified SPE (mSPE) approach for improved peptide yield and analysis sensitivity with LC-MS based peptidomics in terms of time, cost, clogging of the LC-MS column, peptide yield, peptide quality, and number of peptides identified by each method. Expense and time requirements were comparable for both SPE and mSPE, but more interfering contaminants from the urine matrix were evident in the SPE preparations (e.g., clogging of the LC-MS columns, yellowish background coloration of prepared samples due to retained urobilin, lower peptide yields) when compared to the mSPE method. When we compared data from technical replicates of 4 runs, the mSPE method provided significantly improved efficiencies for the preparation of samples from urine (e.g., mSPE peptide identification 82% versus 18% with SPE; p = 8.92E-05). Additionally, peptide identifications, when applying the mSPE method, highlighted the biology of differential activation of urine peptidases during acute renal transplant rejection with distinct laddering of specific peptides, which was obscured for most proteins when utilizing the conventional SPE method. In conclusion, the mSPE method was found to be superior to the conventional, standard SPE method for urine peptide sample preparation when applying LC-MS peptidomics analysis due to the optimized sample clean up that provided improved experimental inference from the confidently identified peptides.     

A procedure for the small-scale isolation of plant RNA suitable for RNA blot analysis

A small-scale method for the isolation of total RNA from plant tissue is described. The method provides RNA of suitable quantity and quality from 0.2 g fresh tissue for the detection of mRNA species by RNA blot analysis. The entire procedure is adapted to 1.5-ml microfuge tubes and takes less than 5 h. This method is well suited for the isolation of RNA from large numbers of samples or from samples of limited quantity.     

An efficient method for explant sterilization for reduced contamination

An improved method for sterilization of explants was tested and found to be suitable for plants with elongated internodes, such as bamboos. Final cutting of the explants into single nodal segments for inoculation was done only after surface sterilization of multi-nodal explants in a stoppered glass measuring cylinder. This minimized penetration of the contaminants and the sterilizing agents into the exposed intercellular spaces and vascular cavities at the cut ends, thereby minimizing their harmful effects. The method was experimented upon three different plants, viz., bamboo, tea and rose. Through this method the number of cultures getting contaminated was substantially reduced as compared to the conventional means where single nodal explants were used, employing identical treatments. Moreover, in this method, the number of cultures showing bud-break also showed a marked increase thereby resulting in a tremendous increase in the percentage of successfully established proliferating cultures.     

4种黑曲霉基因组DNA提取方法的比较

目的:筛选适合提取曲霉DNA的方法.方法:比较2个菌落培养时间段(3d内和10d左右)提取DNA质量的差异;运用氯化苄法、石英砂+CTAB法、Biospin法和微波法分别提取黑曲霉基因组DNA,然后用直接电泳、浓度测定、PCR扩增等方法比较所提DNA的浓度和质量.结果:培养3d内的菌落提取的DNA纯度较高,无需纯化即可用于后续实验;4种方法制备的DNA均可用于PCR等后续实验,其中以石英砂+CTAB法提取的DNA纯度好,产率最高.结论石英砂+CTAB法是一种适用于曲霉DNA提取的简便方法.     

A Rapid Method for Microbial Sample Preparation for the Scanning Electron Microscope

A synthetic aromatic polymer has been used for preparing replicas of different microorganisms. This method of preparing highly concentrated (9.6 k) microbiological samples for scanning electron microscopy was compared with a standard method. The micrographs of the replicated samples are satisfactory. This method is rapid, cost effective and produces good results, especially in the case of spore-forming mycelial microorganisms.     

Express Protocol for Functionalization of Polymer Supports for Oligonucleotide Synthesis

Abstract

A rapid and general one-pot method is described for the attachment of the leader nucleoside onto the polymer supports, suitable for polymer supported oligonucleotide synthesis following oxidation-reduction condensation. The method can also be used for support functionalisation in fully automated DNA synthesizer prior to oligonucleotide synthesis.     

A post-processing method for optimizing synthesis strategy for oligonucleotide microarrays

The broad applicability of gene expression profiling to genomic analyses has generated huge demand for mass production of microarrays and hence for improving the cost effectiveness of microarray fabrication. We developed a post-processing method for deriving a good synthesis strategy. In this paper, we assessed all the known efficient methods and our post-processing method for reducing the number of synthesis cycles for manufacturing a DNA-chip of a given set of oligos. Our experimental results on both simulated and 52 real datasets show that no single method consistently gives the best synthesis strategy, and post-processing an existing strategy is necessary as it often reduces the number of synthesis cycles further.     

New procedure for extraction of ammonium from natural waters for N isotopic ratio determinations

A new method for extracting ammonium from natural waters for N isotopic ratio determination is described. The method employs the conversion of the ammonium nitrogen into indophenol, which is then concentrated onto an octadecylsilane column. The method shows accuracy and precision comparable to those of other methods described in the literature. Some results from field experiments on the Swedish west coast are presented.     

Single-step methods for genomic evaluation in pigs

Genetic evaluation based on information from phenotypes, pedigree and markers can be implemented using a recently developed single-step method. In this paper we compare accuracies of predicted breeding values for daily gain and feed conversion ratio (FCR) in Danish Duroc pigs obtained from different versions of single-step methods, the traditional pedigree-based method and the genomic BLUP (GBLUP) method. In particular, we present a single-step method with an adjustment of the genomic relationship matrix so that it is compatible to the pedigree-based relationship matrix. Comparisons are made for both genotyped and non-genotyped animals and univariate and bivariate models. The results show that the three methods with marker information (two single-step methods and GBLUP) produce more accurate predictions of genotyped animals than the pedigree-based method. In addition, single-step methods provide more accurate predictions for non-genotyped animals. The results also show that the single-step method with adjusted genomic relationship matrix produce more accurate predictions than the original single-step method. Finally, the results for the bivariate analyses show a somewhat improved accuracy and reduced inflation of predictions for FCR for the two single-step methods compared with the univariate analyses. The conclusions are: first, the methods with marker information improve prediction compared with the pedigree-based method; second, a single-step method, contrary to GBLUP, provides improved predictions for all animals compared to the pedigree-based method; and third, a single-step method should be used with an adjustment of the genomic relationship matrix.     

Simple absolute quantification method correcting for quantitative PCR efficiency variations for microbial community samples

Real-time quantitative PCR (qPCR) is a widely used technique in microbial community analysis, allowing the quantification of the number of target genes in a community sample. Currently, the standard-curve (SC) method of absolute quantification is widely employed for these kinds of analysis. However, the SC method assumes that the amplification efficiency (E) is the same for both the standard and the sample target template. We analyzed 19 bacterial strains and nine environmental samples in qPCR assays, targeting the nifH and 16S rRNA genes. The E values of the qPCRs differed significantly, depending on the template. This has major implications for the quantification. If the sample and standard differ in their E values, quantification errors of up to orders of magnitude are possible. To address this problem, we propose and test the one-point calibration (OPC) method for absolute quantification. The OPC method corrects for differences in E and was derived from the ΔΔC(T) method with correction for E, which is commonly used for relative quantification in gene expression studies. The SC and OPC methods were compared by quantifying artificial template mixtures from Geobacter sulfurreducens (DSM 12127) and Nostoc commune (Culture Collection of Algae and Protozoa [CCAP] 1453/33), which differ in their E values. While the SC method deviated from the expected nifH gene copy number by 3- to 5-fold, the OPC method quantified the template mixtures with high accuracy. Moreover, analyzing environmental samples, we show that even small differences in E between the standard and the sample can cause significant differences between the copy numbers calculated by the SC and the OPC methods.     

Rationale for the NCCLS standard H20-T for differential leukocyte counting

The National Committee for Clinical Laboratory Standards (NCCLS) tentative standard for Leukocyte Differential Counting (H20-T) was developed in the United States as a reference method for differential leukocyte counting as well as a method for the evaluation of the performance of differential leukocyte counters. The key features of this document are outlined and the reasons for their inclusion in the standard are given.     

Preparation of Antigen for the Counterimmunoelectrophoretic Test for Plasmacytosis in Mink*

Counterimmunoelectrophoresis as a test method for making the diagnosis of plasmacytosis in mink demands the specific virus antigen. The method for preparation of the antigen according to Cho & Ingram (1972 a, b) with minor modifications is described in details, and results obtained at 62 antigen preparations are presented. In addition an ultrafiltration method is outlined which may be useful as a replacement for ultracentrifugation procedures used in the technique described by Cho & Ingram (1974).