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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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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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New Application for the LightCycler® 480 System: Gene Scanning by High-Resolution Melting

28-08-2007

High-resolution melting (HRM) is a novel, homogeneous, post-PCR method, enabling genomic researchers to analyze genetic variations (SNPs, mutations, methylations) in PCR amplicons. The most important high-resolution melting application is gene scanning - the search for the presence of unknown variations in PCR amplicons prior to or as an alternative to sequencing....

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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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Novel Methods for High-Performance Melting Curve Analysis Using the LightCycler® 480 System

23-08-2007

The LightCycler® Real-time PCR Systems provide fast, accurate, and versatile platforms for genetic variation research. They feature the temperature homogeneity and the optical characteristics re-quired for high-performance melting curve analysis. The plate-based LightCycler® 480 System now facilitates advanced applications in the emerging field of amplicon scanning for new mutations at high resolution....

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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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Epitope Mapping Using the In Vitro Rapid Translation System RTS 100

21-08-2007

The Rapid Translation System (RTS) represents a cell-free system that allows efficient production of protein from linear DNA sequences. This article gives a brief outline of the use of the RTS technology in linear epitope mapping and shows its promise for reducing time-consuming cloning and cell-lysis steps....

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Analysis of the DNA Methylation Patterns at the BRCA1 CpG Island

20-08-2007

Germ-line alterations of the BRCA1 gene confer a lifetime risk of 40% for ovarian cancers and of 40%-80% for ­breast cancers. It is likely that BRCA1 acts as a tumor suppressor gene. BRCA1 involvement in breast cancers does not seem to be restricted to familial cancers. Despite the absence of somatic mutations in the breast tissues, a down regulation of BRCA1 expression is associated with malignancy in human sporadic breast cancers....

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