Nature Methods Application Notes e-UPDATE: 8 May 2012

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8 MAY 2012  Application Notes bring you the latest information about innovative tools and technologies and their applications in the lab. We hope that you will find this service useful and informative and encourage you to sign up for future Updates to ensure that you never miss one!

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FEATURED APPLICATION NOTE A rapid, directional RNA-seq library preparation workflow for Illumina® sequencing www.epibio.com > Most current RNA-seq library preparation methods are time-consuming, multistep processes. We describe a workflow that includes the Ribo-Zero™ and ScriptSeq™ v2 Kits that enables researchers to go from total RNA to cluster-ready RNA-seq libraries in less than 1 d. The RNA-seq libraries produced are virtually free of contaminating ribosomal RNA (rRNA) and provide for directional paired-end and multiplex sequencing on Illumina® sequencing platforms. PDF

		The Cell Comb™ Scratch Assay for Biochemical Analysis of Cell Wound Repair www.millipore.com > The scratch assay is a popular method for the study of cell migration. Scaling up this technique has proved challenging, however, making biochemical analysis of the molecular events mediating wound repair difficult. EMD Millipore's Cell Comb™ scratch assay addresses the need for a simple tool able to create multiple scratch wounds. This article will highlight the use of the Cell Comb™ assay to study cytoskeletal signaling after the wounding of a cell monolayer. PDF

		Assessing mitochondrial dysfunction in primary cardiomyocytes www.seahorsebio.com > This Application Note describes a method for profiling mitochondrial function in cells responding to stress. The mitochondrial profile generated in this way provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity. The results described here suggest that the development of cardiomyocyte injury, in this case caused by an oxidized lipid, increases ATP-linked oxygen consumption, diminishes respiratory efficiency, and depletes the bioenergetic reserve capacity. Seahorse Bioscience recently introduced the XF Cell Mito Stress Test Kit for use with the XF Analyzer, enabling researchers to confidently establish a complete mitochondrial profile with pre-tested and pre-calibrated reagents. PDF

		Measuring fatty acid oxidation in muscle cells www.seahorsebio.com > Altered fatty acid metabolism by skeletal muscle can lead to insulin resistance and the accumulation of lipid in non-adipose tissue. This accumulation plays a critical role in the pathogenesis of chronic diseases including diabetes and heart failure. PDF

		Using Three-dimensional Polystyrene (PS) Scaffolds™ to Produce Tumor Spheroids www.3dbiotek.com > In vitro tumor spheroids are generated using scaffold-free technologies, e.g. hanging drop, agar gel sandwich, and low adhesive surfaces, etc, for drug screening applications. Some of these platforms are currently adapting towards reducing spheroid time formation and increasing high-throughput workflow. Here, we show the use of 3-dimensional (3D) polymer scaffolds to generate multicellular spheroids in an efficient manner. Using 3D Polystyrene (PS) Inserts™, a 3D scaffold with 100% pore interconnectivity, several cancer cell lines were able to produce spheroids as early as 2 days in culture. Further characterization of spheroid formation showed that the number and size of the spheroids appear to be dependent on cell type and scaffold geometry. For example, endometrial cancer cells generated a large pool of small spheroids and osteosarcoma cultured in the scaffold produced a fewer pool of larger spheroids. In conclusion, the 3D PS Insert™ represents a novel platform to generate spheroids for standard drug screening and potential in vitro cancer and stroma co-culture models. In vitro tumor spheroids are generated using scaffold-free technologies, e.g. hanging drop, agar gel sandwich, and low adhesive surfaces, etc, for drug screening applications. Some of these platforms are currently adapting towards reducing spheroid time formation and increasing highthroughput workflow. Here, we show the use of 3-dimensional (3D) polymer scaffolds to generate multicellular spheroids in an efficient manner. Using 3D Polystyrene (PS) Inserts™, a 3D scaffold with 100% pore interconnectivity, several cancer cell lines were able to produce spheroids as early as 2 days in culture. Further characterization of spheroid formation showed that the number and size of the spheroids appear to be dependent on cell type and scaffold geometry. For example, endometrial cancer cells generated a large pool of small spheroids and osteosarcoma cultured in the scaffold produced a fewer pool of larger spheroids. In conclusion, the 3D PS Insert™ represents a novel platform to generate spheroids for standard drug screening and potential in vitro cancer and stroma co-culture models. PDF

		Differential lysis in a single-tube extraction process for accurate forensic profiling www.biotype.de > Sampletype i-sep® DL extraction system was developed for multistage DNA extraction procedures. The novel spin-column system is ideally suitable for DNA extraction procedures involving differential lysis, thus, a potential application to forensic analysis of sexual assault evidence. The extraction system allows for the first time an immunological pre-test using the identical sample. The workflow using Sampletype i-sep® DL is described employing differential lysis of a mixture of epithelium and sperm traces on textile including a pre-test for semen. Sampletype i-sep® has the potential to decrease the forensic DNA analysis backlog of sexual assault cases by circumventing time-consuming conventional differential extraction procedure. PDF

		MAPS – A service-oriented, customizable, multi-purpose LIMS www.ait.ac.at > Summary: MAPS is a LIMS that was developed to be applicable in multiple fields of life sciences (like genomics, genetics, proteomics) and for various applications. It uses an innovative, flexible data model and comes with numerous technologies for adaptation, integration, and extension. In this way, MAPS especially meets the frequently changing requirements of R&D laboratories. Availability and Implementation: MAPS is freely available at http://www.picme.at/maps. It can work with any Hibernate-supported DBMS at the data layer and has been used in routine applications with PostgreSQL 8.3. JBoss 4.2 and SEAM form the application layer, while JSF and ICEfaces are used at the presentation layer. All major current browsers are supported by the system. Supplementary information: Further details about MAPS can be found at http://www.picme.at/maps. PDF

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