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At least 847 records · Page 47Linked to original sources
PubMed2003

Optimizing stringency for expression microarrays.

While several studies have reported methods to optimize expression microarray protocols, none have dealt directly with hybridization wash stringency. We designed a series of experiments to determine the optimal stringency conditions for microarray experiments, using reproducibility and magnitudes of log2 (test/reference) ratio values as measures of quality. Low-stringency wash conditions of cell line hybridizations led to nonspecific binding, resulting in increased intensities, decreased magnitude of ratios, and poor reproducibility. Relatively high-stringency wash conditions were found to give the best reproducibility and large magnitude ratio changes, although increasing the stringency beyond this point led to lower magnitude ratios and poorer reproducibility. The expression levels of the ERBB2 oncogene in the BT474 versus MCF7 cell lines showed that high-stringency wash conditions gave the best agreement with real-time quantitative PCR, although the magnitude of the changes by microarray was smaller than for real-time quantitative PCR. Analysis of a series of cell lines washed at the optimized stringency indicated that the rank order of relative expression levels for ERBB2 microarray clones agreed well with the rank order of ERBB2 levels, as measured by quantitative PCR. These results indicate that the optimization of stringency conditions will improve microarray reproducibility and give more representative expression values.

Artifacts↗
PubMed1995

[Value of turbo-spin-echo sequences in cerebral magnetic resonance tomography in children].

AIM: The value of turbo-spin-echo (TSE) sequences was compared with conventional spin-echo (SE) and inversion-recovery (IR) sequences for cerebral MRT in 70 children at 0.5 T and 1.5 T. In addition we evaluated whether proton weighted sequences (PD) were diagnostically important and in what proportion of cases. METHOD: Conventional T1 and T2 weighted SE and T2 weighted TSE sequences were used in all children. An IR sequence was performed in 39 patients. The various sequences were analysed semiquantitatively with regard to image quality, artifacts and the demonstration of normal and anatomical structures and pathological findings. RESULTS: By any criteria, TSE sequences were superior to conventional T2 weighted SE sequences at 0.5T and 1.5 T, requiring a shorter examination time (35-53%). In 8.6% the pathological finding was best seen on PD-SE sequences (5 glial scars, 1 tumor). CONCLUSION: Although TSE sequences are better than T2 SE sequences with regard to image quality and the demonstration of abnormalities, conventional double-echo SE sequences (with PD and T2 weighted images) cannot be entirely replaced by T2 weighted TSE sequences in children.

Adolescent↗
PubMed2006

Rat toxicogenomic study reveals analytical consistency across microarray platforms.

To validate and extend the findings of the MicroArray Quality Control (MAQC) project, a biologically relevant toxicogenomics data set was generated using 36 RNA samples from rats treated with three chemicals (aristolochic acid, riddelliine and comfrey) and each sample was hybridized to four microarray platforms. The MAQC project assessed concordance in intersite and cross-platform comparisons and the impact of gene selection methods on the reproducibility of profiling data in terms of differentially expressed genes using distinct reference RNA samples. The real-world toxicogenomic data set reported here showed high concordance in intersite and cross-platform comparisons. Further, gene lists generated by fold-change ranking were more reproducible than those obtained by t-test P value or Significance Analysis of Microarrays. Finally, gene lists generated by fold-change ranking with a nonstringent P-value cutoff showed increased consistency in Gene Ontology terms and pathways, and hence the biological impact of chemical exposure could be reliably deduced from all platforms analyzed.

Animals↗
PubMed2002

Screening for stable mutants with amino acid pairs substituted for the disulfide bond between residues 14 and 38 of bovine pancreatic trypsin inhibitor (BPTI).

We have developed a screening method to identify stable protein mutants from a large number of sequences using a cellular quality control system. This method was used to screen amino acid pairs substituted for the disulfide (S-S) bond between residues 14 and 38 of bovine pancreatic trypsin inhibitor. The mutants selected could be divided into two groups: one with mutation C14G and the other with mutation C38V. Although each mutation did not fully compensate for the destabilizing effect of removal of the S-S bond, these mutants have midpoint temperatures of thermal unfolding that are 12-17 degrees C higher than that of the C14A/C38A mutant. This fact indicates that these mutations are better substitutions for the S-S bond than C14A/C38A. The C14G mutants inhibited trypsin more strongly at 37 degrees C than did the C14A/C38A mutant, although bulky amino acids at position 14 largely diminished the inhibitory activity of the C38V mutants. Thermodynamic analysis indicated that the enthalpy of unfolding of the C14G and C38V mutant groups differed considerably, which suggests different stabilizing mechanisms in these two groups. Because renaturation of S-S bonds is often difficult in the large scale production of proteins, this method should provide a useful tool with which to increase the production of recombinant proteins by eliminating S-S bonds with minimum concomitant stability loss.

Amino Acid Sequence↗
PubMed2026

The Tor pathway, ribosome concentration, and wobble decoding mediate inhibitory effects of the Leu-Pro CUC-CCG codon pair in Saccharomyces cerevisiae.

Translation elongation and efficiency are modulated by the genetic code, with reduced translation efficiency and slow translation caused by 17 inhibitory codon pairs in the yeast Saccharomyces cerevisiae Nine of these inhibitory pairs are functionally important as they are disproportionately strongly conserved within orthologous genes in Saccharomyces sensu stricto For three pairs, including CGA-CGA, inhibition is triggered by ribosome collisions followed by known quality control responses, but the mechanisms by which nine other pairs cause inhibition are unknown. Here, our examination of the molecular basis of inhibition by one such pair, the highly conserved Leu-Pro CUC-CCG codon pair, yielded four findings. First, inhibition is mediated by tRNALeu(UAG), which decodes CUC by a U•C wobble interaction and effectively competes with the nonessential Watson-Crick base-pairing tRNALeu(GAG) Second, despite nearly universal conservation of U33 in tRNAs, the C33 alteration in tRNALeu(GAG) does not significantly impair its function. Third, inhibition likely is mediated by ribosome collisions, as many suppressors bear mutations known or predicted to reduce ribosome concentration, and as local reduction in ribosome concentration suppresses inhibition. Thus, differences between CUC-CCG and CGA-CGA inhibition likely occur downstream from ribosome collisions. Fourth, we find a link between the metabolic state and CUC-CCG inhibition, as we find six suppressor mutations in SCH9, a downstream effector of TORC1 that mediates ribosome production. As Sch9 is inactive during starvation, causing reduced ribosome concentration, one biological function of inhibitory pairs may be to mediate a change in relative expression during starvation conditions.

Ribosomes↗
PubMed2006

Three-color cDNA microarrays with prehybridization quality control yield gene expression data comparable to that of commercial platforms.

Despite their lower cost and high content flexibility, a limitation of in-house-prepared arrays has been their susceptibility to quality control (QC) issues and lack of QC standards across laboratories. Therefore, we developed a novel three-color array system that allows prehybridization QC as well as the Matarray software to facilitate acquisition of accurate gene expression data. In this study, we compared performance of our rat cDNA array to the Affymetrix RG-U34A and Agilent G4130A arrays using 2,824 UniGenes represented on all three arrays. Before data filtering, poor interplatform agreement was observed; however, after data filtering, differentially expressed UniGenes exhibited correlation coefficients of 0.91, 0.88, and 0.92 between the Affymetrix vs. Agilent, Affymetrix vs. cDNA, and Agilent vs. cDNA arrays, respectively. The Affymetrix, Agilent, and cDNA arrays agreed well with quantitative RT-PCR conducted on 42 UniGenes, yielding correlation coefficients of 0.90, 0.90, and 0.96, respectively. Each platform underestimated ratios relative to quantitative RT-PCR, possessing respective slopes of 0.86 (R2 = 0.81), 0.65 (R2 = 0.81), and 0.70 (R2 = 0.92). Overall, these data show that the combination of our novel technical and analytic approaches yield an accurate platform for functional genomics that is concordant with commercial discovery arrays in terms of identifying regulated genes and pathways.

Analysis of Variance↗
PubMed2004

Improvements in the HbVar database of human hemoglobin variants and thalassemia mutations for population and sequence variation studies.

HbVar (http://globin.cse.psu.edu/globin/hbvar/) is a relational database developed by a multi-center academic effort to provide up-to-date and high quality information on the genomic sequence changes leading to hemoglobin variants and all types of thalassemia and hemoglobinopathies. Extensive information is recorded for each variant and mutation, including sequence alterations, biochemical and hematological effects, associated pathology, ethnic occurrence and references. In addition to the regular updates to entries, we report two significant advances: (i) The frequencies for a large number of mutations causing beta-thalassemia in at-risk populations have been extracted from the published literature and made available for the user to query upon. (ii) HbVar has been linked with the GALA (Genome Alignment and Annotation database, available at http://globin.cse.psu.edu/gala/) so that users can combine information on hemoglobin variants and thalassemia mutations with a wide spectrum of genomic data. It also expands the capacity to view and analyze the data, using tools within GALA and the University of California at Santa Cruz (UCSC) Genome Browser.

Databases, Genetic↗
PubMed2005

Quality assessment of microarrays: visualization of spatial artifacts and quantitation of regional biases.

BACKGROUND: Quality-control is an important issue in the analysis of gene expression microarrays. One type of problem is regional bias, in which one region of a chip shows artifactually high or low intensities (or ratios in a two-channel array) relative to the majority of the chip. Current practice in quality assessment for microarrays does not address regional biases. RESULTS: We present methods implemented in R for visualizing regional biases and other spatial artifacts on spotted microarrays and Affymetrix chips. We also propose a statistical index to quantify regional bias and investigate its typical distribution on spotted and Affymetrix arrays. We demonstrate that notable regional biases occur on both Affymetrix and spotted arrays and that they can make a significant difference in the case of spotted microarray results. Although strong biases are also seen at the level of individual probes on Affymetrix chips, the gene expression measures are less affected, especially when the RMA method is used to summarize intensities for the probe sets. A web application program for visualization and quantitation of regional bias is provided at http://www.discover.nci.nih.gov/affytools. CONCLUSION: Researchers should visualize and measure the regional biases and should estimate their impact on gene expression measurements obtained. Here, we (i) introduce pictorial visualizations of the spatial biases; (ii) present for Affymetrix chips a useful resolution of the biases into two components, one related to background, the other to intensity scale factor; (iii) introduce a single parameter to reflect the global bias present across an array. We also examine the pattern distribution of such biases and conclude that algorithms based on smoothing are unlikely to compensate adequately for them.

Artifacts↗
PubMed2003

Optimized sensitivity of allele-specific PCR for prenatal typing of human platelet alloantigen single nucleotide polymorphisms.

PCR using sequence-specific primers (PCR-SSP) is widely employed for the genotyping of single nucleotide polymorphisms (SNPs) in both routine diagnosis and medical research. The human platelet alloantigens (HPAs) represent SNPs in platelet-specific glycoproteins, and HPA-1, -2, -3, and -5 are the most relevant in immunohematology. In most protocols, the respective HPA-SNPs are analyzed in allele-specific reactions, each with at least 100 ng DNA. In many cases, prenatal HPA typing in the diagnosis of neonatal alloimmune thrombocytopenia is often limited by the restricted amounts of fetal DNA that are obtainable. We developed a novel PCR-SSP technique to achieve accurate HPA genotypes using only 1 ng DNA per reaction. The concentration of HPA-specific primers was increased to 1 microM each and exhibited a higher sensitivity compared to a commercial PCR-SSP kit. The modified PCR-SSP technique enabled the identification of fetal HPA genotypes using only 0.5 mL amniotic fluid (from week 16 of gestation) and from a maternal plasma sample (from week 38 of gestation). The principle of the modified PCR-SSP technique may also be applied for the genotyping of other SNPs from limited amounts of DNA.

Amniocentesis↗
PubMed1994

Automation and quality control in the coagulation laboratory.

Hemostasis is a balance between complex interactions of directly opposing systems (coagulation and fibrinolysis) with seemingly unrelated systems at both the enzymatic and cellular levels (platelets, endothelium, leukocytes). It should not be surprising that coagulation disorders often accompany many different disease states. Because the function of each protein involved in coagulation is now better defined, newer methodologies have been developed to assay them. In this regard, the hemostasis laboratory can take a two-step approach to diagnosis: global screening and targeted analysis. Several new global test systems provide more detailed, quantitative, and more physiologically relevant evaluations than earlier assays allowed. In addition, individual enzymes, inhibitors, cellular release products, and low molecular weight products of activation reactions (molecular markers) can now be measured in sensitive, specific assays. With this new perspective, genetic predisposition to pathologic hemostatic conditions can be identified through molecular biology and can be identified during the early stages of disease (i.e., at subclinical stages before major pathologic complications are established), and more specifically targeted prophylactic, as well as therapeutic drug interventions, can be administered. The molecular markers of hemostatic activation that can be assessed by various immunochemical methods provide very early evidence of thrombotic, fibrinolytic, or platelet-involved aberrations. Technological advances in methodology and instrumentation have changed the scope of all clinical laboratories but, in particular, that of the coagulation laboratory. The dramatic growth and development have resulted from the influences of clinical chemistry, clinical immunology, pharmacology, biochemistry, and biotechnology. Analytical instruments for use in hemostatic testing go beyond plasma or whole blood clot-based readers, platelet aggregometers, and microscopes to a range of automated, discrete, chemistrylike analyzers, spectrophotometers, microliter ELISA systems, RIA systems, and multiprobe instruments designed to measure simultaneously the different assay end-points of colorimetric and clotting assays and flow cytometers. Instruments are designed for batch processing of single tests on multiple samples or multiple test panels on a single sample. Versatility ranges from instruments that measure only the final reaction solution, by end-point or kinetic analysis, to instruments that automatically pipet reagents and sample, incubate, and analyze the reaction for truly walk-away assay performance. Typically, a wider range of assays are available in automated laboratories as opposed to laboratories performing manual assays.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed2026

scGPA: an LLM-assisted workflow for directional virtual gene perturbation analysis from single-cell transcriptomes.

BACKGROUND: Existing virtual perturbation methods can often infer directional changes by comparing predicted post-perturbation expression profiles with control cells. However, workflows that directly return direction-specific downstream candidate genes together with confidence scores, evidence support and interpretable summaries remain limited. We developed scGPA, an LLM-assisted workflow system for directional single-cell virtual gene perturbation analysis. METHODS: scGPA starts from raw single-cell RNA sequencing data and performs quality control, normalization, dimensionality reduction, clustering and cell-group selection. It then constructs cell-group-specific wild-type regulatory networks using repeated subsampling, principal component regression (PCR)/Ridge-based network inference and CP tensor denoising. Based on these networks, scGPA simulates dose-aware virtual knockdown of the target gene and applies signed perturbation propagation to estimate the magnitude and direction of downstream transcriptional responses. LLM assistance is used for marker-based cell-type annotation, evidence-guided candidate prioritization and user-facing biological summarization. RESULTS: We benchmarked scGPA across five public Perturb-seq datasets and compared its performance with GEARS, scGPT and a random baseline. The overall correct prediction rate of scGPA was 23.0%, exceeding those of GEARS (20.7%), scGPT (15.1%) and the random baseline (13.6%). These results indicate that scGPA achieved a higher correct prediction rate than the two comparator models and the random baseline. We subsequently evaluated scGPA using a public osteosarcoma single-cell dataset and performed qRT-PCR validation in 143B osteosarcoma cells. Among genes with significant experimental changes, scGPA achieved a directional concordance of 76.9%. When all tested downstream genes were counted, 37.0% were directionally correct, 51.9% showed no significant change and 11.1% changed in the opposite direction. CONCLUSIONS: scGPA provides a practical workflow system for predicting and prioritizing direction-specific downstream transcriptional responses after target-gene perturbation. By integrating single-cell regulatory network inference, signed virtual perturbation and LLM-assisted interpretation, scGPA supports target-gene function inference and downstream mechanistic investigation from single-cell transcriptomic data.

Single-Cell Gene Expression Analysis↗
PubMed2006

The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements.

Over the last decade, the introduction of microarray technology has had a profound impact on gene expression research. The publication of studies with dissimilar or altogether contradictory results, obtained using different microarray platforms to analyze identical RNA samples, has raised concerns about the reliability of this technology. The MicroArray Quality Control (MAQC) project was initiated to address these concerns, as well as other performance and data analysis issues. Expression data on four titration pools from two distinct reference RNA samples were generated at multiple test sites using a variety of microarray-based and alternative technology platforms. Here we describe the experimental design and probe mapping efforts behind the MAQC project. We show intraplatform consistency across test sites as well as a high level of interplatform concordance in terms of genes identified as differentially expressed. This study provides a resource that represents an important first step toward establishing a framework for the use of microarrays in clinical and regulatory settings.

Equipment Design↗
PubMed1988

Functional role of a highly repetitive DNA sequence in anchorage of the mouse genome.

The major portion of the eukaryotic genome consists of various categories of repetitive DNA sequences which have been studied with respect to their base compositions, organizations, copy numbers, transcription and species specificities; their biological roles, however, are still unclear. A novel quality of a highly repetitive mouse DNA sequence is described which points to a functional role: All copies (approximately 50,000 per haploid genome) of this DNA sequence reside on genomic Alu I DNA fragments each associated with nuclear polypeptides that are not released from DNA by proteinase K, SDS and phenol extraction. By this quality the repetitive DNA sequence is classified as a member of the sub-set of DNA sequences involved in tight DNA-polypeptide complexes which have been previously shown to be components of the subnuclear structure termed 'nuclear matrix'. From these results it has to be concluded that the repetitive DNA sequence characterized in this report represents or comprises a signal for a large number of site specific attachment points of the mouse genome in the nuclear matrix.

Animals↗
PubMed1990

Sex, sex role identification, and college students' projective drawings.

A study of 109 undergraduates found that sex and sex role identification, as assessed by the Bem Sex Role Inventory (BSRI), were related significantly to sex sequence, sexual differences, and quality of drawing on the Draw-A-Person (DAP). Specifically, males and masculine persons tended to draw males first, female and feminine persons tended to draw females first, and androgynous persons were equally likely to draw a male or a female first. The greater the sexual differentiation of the female human figure drawing, the lower the subject's degree of masculinity. The single best predictor for identifying a female subject was the quality of the female figure drawing. Results were consistent with Machover's hypothesis that drawings of the human figure reveal facets of the subject's self-image.

Adolescent↗
PubMed2004

The many colours of chromodomains.

Local differences in chromatin organisation may profoundly affect the activity of eukaryotic genomes. Regulation at the level of DNA packaging requires the targeting of structural proteins and histone-modifying enzymes to specific sites and their stable or dynamic interaction with the nucleosomal fiber. The "chromodomain", a domain shared by many regulators of chromatin structure, has long been suspected to serve as a module mediating chromatin interactions in a variety of different protein contexts. However, recent functional analyses of a number of different chromodomains revealed an unexpected diversity of interaction targets, including histones, DNA and even RNA. The chromodomains of today seem to have evolved from a common ancestral fold to fulfill various functions in different molecular contexts. Combining information gained from recent functional and structural studies of chromodomains with a bioinformatic classification of their structure could lead to the definition of sequence motifs with predictive quality for chromodomain function.

Animals↗
PubMed2006

A quality assessment survey of SNP genotyping laboratories.

To survey the quality of SNP genotyping, a joint Nordic quality assessment (QA) round was organized between 11 laboratories in the Nordic and Baltic countries. The QA round involved blinded genotyping of 47 DNA samples for 18 or six randomly selected SNPs. The methods used by the participating laboratories included all major platforms for small- to medium-size SNP genotyping. The laboratories used their standard procedures for SNP assay design, genotyping, and quality control. Based on the joint results from all laboratories, a consensus genotype for each DNA sample and SNP was determined by the coordinator of the survey, and the results from each laboratory were compared to this genotype. The overall genotyping accuracy achieved in the survey was excellent. Six laboratories delivered genotype data that were in full agreement with the consensus genotype. The average accuracy per SNP varied from 99.1 to 100% between the laboratories, and it was frequently 100% for the majority of the assays for which SNP genotypes were reported. Lessons from the survey are that special attention should be given to the quality of the DNA samples prior to genotyping, and that a conservative approach for calling the genotypes should be used to achieve a high accuracy.

Clinical Laboratory Techniques↗
PubMed1998

Simultaneous multislice acquisition with arterial-flow tagging (SMART) using echo planar imaging (EPI).

Arterial spin tagging techniques have been used to image tissue perfusion in MR without contrast injection or ionizing radiation. Currently, spin tagging studies are performed primarily using single-slice imaging sequences, which are time consuming. This note reports a multislice echo-planar arterial spin tagging technique (Simultaneous Multislice Acquisition with aRterial-flow Tagging, or "SMART"). Multiband RF encoding (Hadamard) is used to provide simultaneous multislice acquisition capability for spin tagging techniques (such as echo planar imaging signal targeting with alternating radio frequency and flow-sensitive alternative inversion recovery). The method is illustrated with a two-slice pulse sequence that was implemented using the FAIR technique to generate two perfusion weighted images simultaneously. Compared with single-slice sequences, this two-slice sequence provided similar image quality, signal-to-noise ratio, and twice the spatial coverage compared with the single-slice technique within the same scan time.

Blood Flow Velocity↗
PubMed2005

An in-gel digestion procedure that facilitates the identification of highly hydrophobic proteins by electrospray ionization-mass spectrometry analysis.

A procedure is described for in-gel tryptic digestion of proteins that allows the direct analysis of eluted peptides in electrospray ionization (ESI) mass spectrometers without the need of a postdigestion desalting step. It is based on the following principles: (a) a thorough desalting of the protein in-gel before digestion that takes advantage of the excellent properties of acrylamide polymers for size exclusion separations, (b) exploiting the activity of trypsin in water, in the absence of inorganic buffers, and (c) a procedure for peptide extraction using solvents of proven efficacy with highly hydrophobic peptides. Quality of spectra and sequence coverage are equivalent to those obtained after digestion in ammonium bicarbonate for hydrophilic proteins detected with Coomassie blue, mass spectrometry-compatible silver or imidazole-zinc but are significantly superior for highly hydrophobic proteins, such as membrane proteins with several transmembrane domains. ATPase subunit 9 (GRAVY 1.446) is a membrane protein channel, lipid-binding protein for which both the conventional in-gel digestion protocol and in solution digestion failed. It was identified with very high sequence coverage. Sample handling after digestion is notably simplified as peptides are directly loaded into the ESI source without postdigestion processing, increasing the chances for the identification of hydrophobic peptides.

Adenosine Triphosphatases↗