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Molecular Probes® NanoOrange® Assay Performed on BMG LABTECH FLUOstar OPTIMA Microplate Reader

08-05-2008

The field of proteomics has expanded dramatically in recent years with research on a whole host of organisms. Techniques such as two dimensional difference gel electrophoresis (2D DIGE) require the accurate quantitation of protein, as up to three labelled samples can be loaded on a single gel for comparison. Assay methods for determining protein quantitation include absorbance at 280 nm(1), the Bradford assay(2), Lowry assay(3), BCA method(4) and more sensitive assays such as Fluoroprofile® (Sigma-Aldrich) and NanoOrange® (Molecular Probes®)5()....

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Enzymatic Protein Digest for Mass-spectrometric Analysis

26-09-2007

The main focus in proteomic studies is on the identification of proteins in given biological samples. Proteins isolated and separated from a given sample (e.g., whole cell lysates, blood or tissue, protein complexes) by immunoprecipitation or affinity chromatography or two-dimensional electrophoresis must be proteolytically cleaved in the course of sample preparation for identification and characterization. A reproducible cleavage pattern of digested proteins is a prerequisite for the unambiguous identification of these proteins by mass spectrometry (MS)....

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High Pure PCR Cleanup Micro Kit - Novel Flexibility for Nucleic Acid Purification

19-09-2007

Manual nucleic acid isolation with High Pure products is long approved by the scientific community. The product portfolio includes kits for isolation of genomic RNA and DNA, plasmid DNA as well as purification of reaction products. Key goal for all products is to offer optimal solutions suited for many different sample materials in order to reduce the number of different nucleic acid isolation kits necessary in modern molecular biology labs....

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Genotypes in Smokers: Correlations with Smoking Behavior

12-09-2007

Smoking behavior is influenced by both genetic and environmental factors. Moreover, smoking initiation and smoking persistence have a heritability of at least 50%. A large body of work has been dedicated to associating multiple genetic markers of neurobiological pathways previously linked to addiction and smoking such as the central dopaminergic, serotoninergic, and nicotinergic pathways with different aspects of smoking behavior. In order to investigate the feasability of future genetic analyses, we genotyped 14 SNPs in 288 samples of addicted smokers using the LightCycler® 480 System with HybProbe assays....

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Real-Time PCR Quantification of Plant miRNAs Using Universal ProbeLibrary Technology

29-08-2007

MicroRNAs (miRNAs) are naturally occurring, highly conserved families of transcripts (18-25 nt in length) that are processed from larger hairpin precursors. Both plant and animal genomes encode miRNAs, and to date, there are over 4000 mature miRNA transcripts annotated. Although miRNAs represent a relatively abundant class of transcripts, their expression levels vary greatly among species and tissues. Less abundant miRNAs routinely fail detection using technologies such as cloning, northern hybridization and microarray analysis. Here, we present a real-time quantification method for accurate, sensitive, and economical detection of plant miRNAs based upon the stem-loop RT primer approach using an miRNA-specific primer coupled with a stem-loop specific Universal ProbeLibrary (UPL) probe. This approach represents a highly specific, sensitive, and easy-to-apply system to detect miRNAs....

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The Universal ProbeLibrary - a Versatile Tool for Quantitative Expression Analysis in the Zebrafish

29-08-2007

The teleost zebrafish is a widely used model organism for the study of developmental and disease-related processes in vertebrates, including humans. Understanding these processes at a molecular and cellular level frequently requires the estimation of gene expression levels. Quantitative real-time PCR (qPCR) is an important tool towards this goal but has, until now, been relatively neglected in zebrafish. Here, we describe the comparison of the Universal ProbeLibrary (UPL)-based qPCR to a conventional SYBR Green I-based qPCR assay using embryonic zebrafish cDNA. We found that the Universal ProbeLibrary-based assay shows equal or better performance compared with the SYBR Green I assay....

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Real-Time PCR Quality Control for Gene Expression

Profiling Using the LightCycler® 480 System

28-08-2007

Quantitative real-time PCR (qPCR) has become the de facto standard for nucleic acid quantification. This achievement is due in large part to its sensitivity, specificity, accuracy, large dynamic range of linear quantification, and its speed. The qPCR technology has matured to a ready-to-use commonly available method in most molecular biology laboratories. Nevertheless, the reliability of the final quantification result depends heavily on all elements in the workflow, such as the quality of the input template (integrity and absence of inhibitors), the PCR assay (specificity, efficiency, limit of detection), and normalization strategy (validated reference genes)....

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Quantification of Zea mays mRNAs by Real-Time PCR Using the Universal ProbeLibrary

24-08-2007

The Universal ProbeLibrary (UPL) is a detection system for real-time PCR based on locked nucleic acid (LNA) probes. We tested the efficiency of the UPL for the quantification of maize transcripts on two detection platforms. The results indicate that the UPL is an attractive alternative to assays based on intercalating dyes or conventional probes....

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Quantitative Detection of Legionella pneumophila in Water Samples: Assay Transfer to the LightCycler® 480 Real-Time PCR System

22-08-2007

An assay to detect and quantify Legionella spp. in water samples was successfully transferred from a capillary-based LightCycler® Instrument to the novel multiwell plate-based LightCycler® 480 System. A previously described protocol was easily adapted by modifying the fluorescent label of the detection probe to match the filter set of the LightCycler® 480 Instrument. While reproducibility and analytical sensitivity of the assay were found to be comparable on both systems, the novel plate-based instrument allowed for higher sample throughput and reduced assay time per sample....

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LightCycler® 480 System: High-throughput Gene Expression and Genotyping Analysis - A Performance Study

09-08-2007

The LightCycler® 480 System is a novel platform for rapid gene expression and melting curve-based genotyping for medium- and high-throughput. In order to assess the performance and ease-of-use of the complete LightCycler® 480 System (instrument, software, and reagents) in an academic laboratory setting we scheduled a high-throughput project including gene expression (39 targets in 88 samples = 3,400 expression values) as well as genotyping applications (3,808 genotypes; 14 SNPs in 288 samples). The project, involving five technicians and two graduate students, was accomplished within 15 working days....

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