38 Current white paper of Roche Diagnostics

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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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Efficient Transfection of Primary Human Skeletal Myoblasts Using FuGENE® HD Transfection Reagent

26-09-2007

Transfection of cells is one of the main techniques used to influence gene expression. Most primary cells and human skeletal myoblasts (SkMC) in particular are very difficult to transfect, whereas for cell lines such as C2C12, many suitable transfection reagents and protocols are available. Few publications report successful transfection of human primary myoblasts using non-viral systems [1,2,3]. These methods include cationic lipids such as phosphono­lipids, electroporation, and a combination of liposome and adenoviral associated proteins. But transfection efficiency is low and often is a compromise between toxicity of the reagents and transfection efficiency....

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Real-Time Multiplex PCR of five Different DNA Targets Using the LightCycler® 480 System

19-09-2007

One of the most interesting aspects of real-time PCR based on detection of fluorophoric-labeled oligonucleo­tides, such as Hydrolysis probes, and Molecular Beacons, is the possibility of being able to detect conveniently multiple targets in the same PCR reaction (multiplex PCR). Ideally, a real-time multiplex PCR should be able to detect, differentiate, and provide a quantitative result for many different targets without a single target influencing the detection of one of the others (cross-talk) and without loss of sensitivity. It is evident that due to the limited number of fluorophoric labels available and the significant overlap in their emission spectra, quantification of multiplex reaction products is difficult and often not possible for more than two targets....

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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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Mutation Scanning of the Cytidine Deaminase Gene by High-Resolution Melting Curve Analysis Using the LightCycler® 480 System

12-09-2007

Alexandre Evrard1,2, Caroline Raynal1,2, Jean-Christophe Boyer2, Lionel Le Gallic2, and Serge Lumbroso1,2 1Institut de Génomique Fonctionnelle, Département d'Oncologie Moléculaire et Cellulaire, Montpellier, France, 2Laboratoire de Biochimie, Hôpital Carémeau, CHU Nîmes, France *Corresponding author...

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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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Metagenomics Analysis Using the Genome Sequencer FLX System

05-09-2007

The Genome Sequencer FLX System from 454 Life Sciences and Roche Applied Science is a versatile sequencing platform suitable for a wide range of applications, including de novo sequencing and assembly of genomic DNA, transcriptome sequencing, small RNA analysis, and amplicon sequencing. The Genome Sequencer FLX is built upon 454 Sequencing technology that enables long read lengths and very high single-read accuracy. One application in which the technology is accelerating the field's understanding is metagenomics....

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Experimental Workflow for Fast and Accurate Genotyping of Scrapie-resistant/sensitive Sheep

05-09-2007

Several scientific studies support the claim that natural scrapie is associated with polymorphisms at three codons within the sheep prion protein (PrP) gene. Genotyping of these codons (codon 136 [alanine, valine], 154 [arginine, histidine], and 171 [glutamine, arginine, histidine]) might enable breeding of sheep flocks with high resistance to scrapie. Fast and reliable high-throughput genotyping of sheep is a major issue, since scrapie is a serious problem in several European countries. To address this, an experimental procedure for DNA preparation and PCR-based detection of codon 136, 154, and 171 polymorphisms was established....

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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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