Nature Methods Application Notes e-UPDATE: 12 June 2012

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12 JUNE 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 transposable approach to RNA-seq from total RNA www.epibio.com > The rapid progress of RNA-seq technology requires the development of simple, versatile tools that competently reflect RNA levels in the cell. We have developed the TotalScript™ RNA-Seq Kit, a new, transposome-based directional library prep method suitable for a variety of RNA sample types and a range of input. TotalScript libraries exhibit high complexity, ∼97–99% directionality and low rRNA contamination. The method is scalable and robust, allowing input amounts of ∼100 pg of poly(A)+ RNA or ∼5 ng of total RNA. 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

		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

		Comparative analysis of three bovine genomes www.clcbio.com > Comparative genomics data analysis using Genomics Gateway: The goal is to identify zebu-specific variations which are not present in bison or in taurine (Cosart et al. 2011). Furthermore, we attempt to link the zebu-specific sequence variations to altered pathways using the gene ontology tool built into Genomics Gateway. PDF

		Time savings and improved reproducibility through simple and fast separation of substrate from lysate www.biotype.de > During DNA-analysis sample preparation steps claim a large proportion of the available time. Protocol optimization contributes substantially to increased laboratory productivity. The Sampletype i-sep® SQ extraction system was developed to improve the process of sample preparation by optimizing yield and reproducibility of DNA isolation; as the quantity and quality of genomic DNA extracted from any sample greatly impacts the success of the downstream analysis and the overall quality of the final result. This is particular the case for field specimens, which might be limited in quantity, may be environmentally exposed, and may require purification from difficult substrates containing PCR inhibitors. Proprietary filter material allows the sample lysis and separation of DNA in the same device without any manual transfer of the substrate or additional pipetting steps. The novel column system is ideally suitable for DNA extraction procedures involving a diversity of specimens from both routine clinical and challenging forensic samples. For clinical application the product is CE labeled as in-vitro diagnostic (IVD). PDF

		Efficient and Scalable Protein Expression in Suspension CHO Cells Using the NovaCHOice® Transfection System www.millipore.com > Protein expression in mammalian cells is the method of choice for the production of bioactive proteins displaying appropriate post-translational modifications. A convenient approach to obtain bioactive proteins and shorten the evaluation cycles is to use transient transfection. One of the preferred cells systems for production of bioactive proteins is Chinese Hamster Ovary (CHO) cells that have been adapted for growth in suspension. However, suspension CHO (CHO-S) cells tend to be refractory to transfection using many of the commonly-used transfection reagents. Here we present data on the use of the NovaCHOice® transfection system, consisting of a core reagent and an optional booster, to obtain high levels of protein expression in CHO-S cells. Our data indicate that use of NovaCHOice® transfection system results in high transfection efficiency and high levels of protein expression within 24 h. Our data further indicate that by 48 h and 72 h the proportion of high-expressing cells is significantly increased with very low cellular toxicity. In addition, no medium changes are required post-transfection, thus reducing cell losses and hands-on time. In summary, the NovaCHOice® transfection system is well-suited for transient and stable transfections in CHO-S cells, due to its high transfection efficiency and low toxicity. 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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