Nature Methods Application Notes e-UPDATE: 10 December 2013

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10 DECEMBER 2013  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 High-content analysis of three-dimensional tumor spheroids: investigating signaling pathways using small hairpin RNA www.3dbiomatrix.com > www.ttplabtech.com > http://www.gnf.org > In the past, creating three-dimensional (3D) tumor spheroids that were suitable for high-throughput screening (HTS) was a difficult and often expensive process. We describe how to couple easy, controllable 3D spheroid formation in Perfecta3D® Hanging Drop Plates with the acumen® eX3 high-content imager for rapid, multicolor, whole-well quantification. This process provides researchers with a highly efficient method to achieve physiologically relevant tumor models in an HTS format. PDF

		Rapid ELISA-Based Measurement of Protein Phosphorylation Using RayBio® Phosphorylation ELISA Kits http://www.raybiotech.org > RayBio® Phosphorylation ELISA is a rapid, convenient and sensitive sandwich ELISA for the in vitro measurement of key signaling pathway phosphoproteins in cell or tissue extracts. Over 20 different kits are available for well-studied pathway molecules such as EGFR, Akt, Erk1/2, p38 alpha, Mek1, STAT1, STAT3, and Met. RayBio Phosphorylation ELISA also features site-specific phospho-antibodies which can detect a single phosphorylated residue (for example, Tyr1086 of EGFR). This method is an improvement on traditional labor-intensive immunoblotting protocols that require 2-3 days of processing time. PDF

		The Impact of Transfection Mediated Toxicity - Gene Expression and Cytotoxicity Analysis of Transfection Reagents www.mirusbio.com > While plasmid DNA delivery is a widely used method to study cellular functions of proteins of interest, studies to identify nontoxic gene delivery reagents are limited. With the advent of high-information content technologies, especially RT-qPCR array, it is now possible to identify the gene expression response to a particular cellular insult. This improvement, coupled with the observation that virtually all toxic responses are accompanied by changes in gene expression, suggests that gene expression analysis has the potential to refine the identification of minimal‐toxicity transfection reagent where phenotypic responses such as altered morphology is not immediately evident. Consequently, we conducted an integrative study to explore the conventional toxicological endpoints and to identify the minimal-transcriptomic effects of TransIT®-LT1, TransIT®-2020 and Lipofectamine® 2000 Transfection Reagents using quantitative reverse transcriptase PCR (RT-qPCR) array and pathway analysis software. PDF

		Automation of ChIP-Seq Library Preparation for Next Generation Sequencing on the epMotion®5075 TMX http://www.eppendorfna.org > ChIP-Seq library preparation can be a challenging procedure to automate because of its low ChIP DNA input. This Application Note describes the successful automation of Illumina™ ChIP-Seq library preparation on the Eppendorf® epMotion 5075 TMX, using the KAPA High-Throughput Library Preparation Kit from KAPA Biosystems®. Size-selected libraries were prepared from as little as 1 ng of fragmented ChIP DNA. To obtain the recommended 100 ng of library material for sequencing on the Illumina Genome AnalyzerIIx, only 18 amplification cycles were needed for 1 ng of input DNA, or 9 cycles when starting library construction with 10 ng of ChIP DNA. PDF

		Molecular indexing for improved RNA-Seq analysis http://www.biooscientific.org > In this application note, Bioo Scientific Corporation and Cellular Research introduce a new kit for high precision gene expression analysis by RNA-Seq. We demonstrate that the NEXTflex™ qRNA-Seq™ Kit can be directly substituted into paired-end library preparation without affecting conventional RNA-Seq analysis methods. This new kit efficiently generates libraries equivalent to standard RNA-Seq libraries with sample barcodes, but with the added feature of molecular indexing. The value of using the added molecular indexing feature depends upon experimental design, data objectives, and sequencing depth for a given library complexity. PDF

		Trypsin/Lys-C protease mix for enhanced protein mass spectrometry analysis www.promega.com > Traditionally, trypsin and Lys-C proteases are used in combination to digest proteolytically resistant proteins. Here we describe use of Trypsin/Lys-C Mix for general digestion needs. Working simultaneously under conventional non-denaturing trypsin digestion conditions, Trypsin and Lys-C enhance proteolysis and provide multiple positive effects on protein mass spectrometry analysis including increased number of identified peptides and proteins, higher analytical reproducibility and more accurate protein quantitation. Effectively, by supplementing trypsin with Lys-C, we create improved trypsin. PDF

		A Novel Molecular Approach for Measuring Viral Load www.abionline.com > Advances in molecular techniques have initiated a revolution in the field of infectious diseases. Molecular assays are now routinely used to measure viral load. Many of these assays require a critical nucleic acid extraction step, followed by quantitation of recovered DNA. Extraction methods, however, inherently suffer from inefficiencies in individual steps and loss of nucleic acid during the extraction process. While there are many different methods and technologies available for nucleic acid extractions, none of these include appropriate controls to monitor extraction efficiency and loss of DNA. To ensure reliable and reproducible results, there is a need for appropriate extraction controls to validate nucleic acid extraction methods, and standards for nucleic acid quantitation. PDF

 

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