Nature Methods Application Notes e-UPDATE: 7 August 2012

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7 AUGUST 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

		Qualitative and Quantitative Characterization of the Metabolome, Lipidome, and Proteome of Human Hepatocytes www.waters.com > A label-free multi-omics approach has been applied for the analysis of the transfected human hepatocyte cells by implementing LC/HDMS[E], providing both qualitative and quantitative information within a single experiment. PDF

		RNA Extraction and Small RNA Library Production for Next Generation Sequencing www.biooscientific.com > To maximize the information that can be obtained from the global profiling of small RNAs using NGS, robust protocols are needed for RNA extraction and for preparing the RNA for sequencing. The NEXTprep™ Small RNA Isolation Kit has been optimized and validated for the extraction of small RNA from tissue for use in next generation sequencing (NGS) library preparation. This whitepaper includes methods and images that will be useful for researchers who are new to the RNA-seq field, to benchmark their own data, thus ensuring the quality of their own samples is sufficient for successful use in small RNA-seq applications. 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

		LentiBrite™ Lentiviral Biosensors for Fluorescent Cellular Imaging: Analysis of Autophagosome Formation www.millipore.com > Expression of genetically-encoded fluorescently-tagged proteins has widely been employed for real-time visualization of cellular behavior and trafficking. Prepackaged, ready-to-use, high-titer lentiviral particles (which we have termed "lentiviral biosensors") encoding GFP- or RFP-tagged proteins are a convenient, robust solution for fluorescent imaging of transduced cells. Compared to other nonviral transfection methods, lentiviral transduction, in many cases, offers higher transfection efficiency and more homogeneous protein expression, particularly for traditionally hard-to-transfect primary cell types. Lentiviral biosensors are ideal for use with fixed and live cell fluorescent microscopy, and are non-disruptive towards cellular function. GFP- or RFP-protein localization matches well with antibody-based immunostaining and demonstrates altered patterns of expression upon treatment with modulators of cell function and phenotype. Lentiviral biosensors provide a broadly effective, convenient method for visualization of cell behavior under a variety of physiological and pathological treatment conditions, in both endpoint and real-time imaging modalities. In this study, we focus on lentiviral biosensors containing GFP-LC3 and RFP-LC3 for the study of autophagosome formation. 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

		Expresso® CMV system: effortless mammalian expression cloning www.lucigen.com > The Expresso® systems dramatically increase the speed and efficiency of target gene cloning and protein expression. With Expressioneering™, PCR products are cloned instantly and directionally into pre-processed mammalian expression vectors without sample cleanup or enzyme treatment. PDF

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