Nature Methods Application Notes e-UPDATE: 14 October 2014

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14 October 2014  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 Chromatrap® 96: a new solid-state platform for high-throughput ChIP www.chromatrap.com > Chromatrap® 96 (C96) is a high-throughput analysis platform that profiles up to 96 transcription factors and epigenetic modifications simultaneously in less than 1 d. It enables sensitive, selective and reproducible target amplification with excellent signal-to-noise ratios, even from samples as small as 0.1 μg. Compatible with automated handling, C96 allows simultaneous investigation of parallel epigenetic landscapes, offering unprecedented assay flexibility and speed. PDF

		Using PrimeScript™ Reverse Transcriptase for Quantification of shRNA Knockdown of a Low Abundance Target www.clontech.com > PrimeScript Reverse Transcriptase is a high efficiency enzyme that is ideal for preparing cDNA for the analysis of low abundance transcripts. In this experiment, Cdc14A mRNA, which is expressed at low levels, was quantified in mouse embryonic fibroblast (MEF) cells that had been transduced with either a control shRNA or a Cdc14A-specific shRNA. The PrimeScript RT Reagent Kit (Perfect Real Time) (Cat. #RR037A)and a reverse transcriptase from another supplier (Company I) were used to generate cDNA templates for measuring Cdc14A expression by real-time PCR using SYBR® Green detection. PDF

		CellTiter-Glo® 3D: A Sensitive, Accurate Viability Assay for 3D Cell Cultures www.promega.com > The CellTiter-Glo® 3D Cell Viability Assay is designed for determining cell viability in 3D microtissue spheroids. The assay reagent penetrates large spheroids and has increased lytic capacity—allowing more accurate determination of viability compared to other assay methods. Based on the same chemistry as the classic CellTiter-Glo® Assay, this new 3D assay reagent measures ATP as an indicator of viability, and generates a luminescent readout that is much more sensitive than absorbance or fluorescence-based methods. In addition, the assay protocol is simple and fast, giving results in as little as 30 minutes. PDF

		One Solution for Cloning and Mutagenesis: In-Fusion®HD Cloning Plus www.clontech.com > In this application note, we present a single, convenient system for both cloning and site-directed mutagenesis including deletions, base substitutions, or insertions. In-Fusion HD Cloning Plus is sequence independent, seamless, directional, flexible enough to use with any vector and allows over 95% cloning efficiency to be consistently achieved. Mutagenesis can be carried out by combining the strength of the In-Fusion®HD with inverse PCR. PDF

		Suitability of Medicyte's upcyte® cells for in vitro formation of liver bud-like organoid liver structures www.medicyte.com > upcyte® cells are non-malignant cell strains derived from primary human cells that underwent targeted genetic modification (upcyte® process) resulting in transiently proliferating cells that can undergo up to 40 population doublings. When contact-inhibited upcyte® cells possess the typical morphology of mature cells and show many of the cell type specific characteristics. Formation of three-dimensional structures resembling embryonic liver buds in vitro has recently been described using stem cell derived cell types (Takebe et al., 2013). Such structures can be transplanted into animals but may also prove useful for ex vivo drug toxicity testing systems to more closely resemble the in vivo situation as compared to conventional two-dimensional (monolayer) cell culture models. In this article we present a protocol using a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) that form liver buds in vitro which can be cultured for up to 20 days or even longer. These organoid, self-organized liver-like structures contain living cells showing typical functional characteristics of liver cells, e.g. Rifampicin-induced induction of Cytochrome P450 3A4 (Cyp3A4) activity. PDF

		Chemiluminescent Westerns: How film and photochemistry affect experimental results www.licor.com > Since the 1970s, enhanced chemiluminescence has been used to detect proteins on Western blots. Secondary antibodies are labeled with the horseradish peroxidase (HRP) enzyme, which oxidizes the luminol-based chemiluminescent substrate and causes it to transiently produce light at ~428 nm. This sensitive, reliable detection chemistry is typically documented by exposure of the blot to x-ray film, which darkens in response to the emitted light. Signal intensity is determined by the number of HRP molecules reacting with substrate. Chemiluminescent blots are often analyzed by visual assessment of band intensities. This method is sufficient to confirm presence or absence of a signal, or to compare bands of substantially different intensities. For more detailed analysis, film images may be digitized and relative band intensifies measured by densitometry. PDF

		Randomized Adapters for Reducing Bias in Small RNA-Seq Libraries http://www.biooscientific.org > The past decade has seen an explosion of interest in cataloging the small RNA repertoires of animal and plant species, and in understandingthe biological function of small RNAs. Small RNAs include not only microRNAs, but also piRNAs and other types of endogenous small RNAs.Distinguishing closely related small RNAs is difficult using microarray- and qPCR-based approaches, since imperfectly matched small RNAs may still be able to hybridize to PCR primers or immobilized probes. These considerations have led to the realization that next generation sequencing (NGS) is the most practical method for large-scale small RNA studies that aim to identify and enumerate small RNAs in various species and tissues. NGS offers advantages of sensitivity, specificity, and the ability to maximize data acquisition and minimize costs by using multiplex strategies to allow many samples to be sequenced simultaneously. PDF

 

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