Nature Methods Application Notes e-UPDATE: 9 April 2013

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9 APRIL 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 Discovery and quantification of transcript variants with SQUARE™ mRNA-Seq www.lexogen.com > The majority of RNA-seq library preparation protocols quantify the expression of genes without taking into account the numerous transcript variants which contribute to this overall expression. SQUARE mRNA-seq uses a selective matrix to separate transcript variants and to enable the assembly and quantification of individual transcripts. The protocol is highly specific for full-length, polyadenylated mRNA, and transcript hypotheses are supported by directional transcription start and polyadenylation site tagging. PDF

		Capture vs Coupling of an Antibody Using a Reichert SR7500DC Surface Plasmon Resonance (SPR) System www.ReichertSPR.com > Amine coupling is often the first method a person uses to attach a ligand to an SPR slide. The covalent attachment via free primary amines on a ligand results in random attachment since there are usually multiple attachment sites on the protein. The coupling is done at a pH that is 1-2 units below the pI of the ligand. This facilitates electrostatic attraction to the attachment surface. A second way to attach a ligand to an SPR slide is via non-covalent capture methods. Non-covalent capture methods are typically used to attach ligands to an SPR surface in instances where the ligand cannot withstand the lower pH needed for covalent coupling or when a ligand needs to be attached in a more oriented manner. This approach is applicable to a variety of types of samples. We provide examples of three different approaches to coupling the monoclonal antibody Anti-Human Serum Albumin (HSA) IgG to an SPR slide in this Application Note. The first example is direct amine coupling. The second example is covalently coupling NeutrAvidin to a slide surface and then Capturing biotinylated Anti-HSA. A third example is where Goat Anti-Mouse Fc IgG is amine coupled to a slide surface and then used to capture monoclonal Anti-HSA IgG. In all three cases, antigen binding (HSA) to the Anti-HSA is followed over a series of concentrations and a KD value is determined. PDF

		Effect of microRNA GC Content on RNA Purification Efficiency www.norgenbiotek.com > Unlike messenger RNA and large RNA species, a slight change in base composition in microRNA (miRNA) will drastically alter the molecules GC content and its binding preference to targets. It has been commonly assumed that standard RNA isolation methods work equally well for all miRNAs without bias. In this study, we have shown that GC content does indeed bias purification efficiency in most phenol-based methods tested, and that the best non-biased isolation product is the Silicon Carbide-based technology from Norgen Biotek. PDF

		3D dual-color PALM/dSTORM imaging of centrosomal proteins with nanometric resolution using MicAO 3DSR www.imagine-optic.com > Photoactivation localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) are becoming routine methods in biological imaging with optical resolution beyond the light diffraction barrier. We present MicAO 3DSR – the first adaptive optics device which introduces the three dimensional imaging capability for PALM/STORM. This apparatus, designed by Imagine Optic, contains the wave-front sensor and deformable mirror. With the help of these components MicAO 3D-SR corrects various types of aberrations, induced by optical elements inside the microscope and by the biological sample. MicAO 3D-SR optimizes the Point Spread Function (PSF) of the microscope and consequently improves the lateral localization precision of the PALM/STORM setup by 40%. In addition to that, MicAO 3D-SR brings in the ability to image fluorescent molecules in 3D. With its deformable mirror, it introduces controlled perfect astigmatism and allows us to precisely locate the position of fluorescing molecule in all three dimensions. In this application note we demonstrate the localization precision of PALM/STORM method when it is operated together with MicAO 3D-SR by 3D dual-color imaging of two proteins in centrosome – the cellular organelle which dimensions lye beyond the diffraction limit of 250x250x500 nm. PDF

		The F.A.S.T.™ Gene Modulating System www.genetargeting.com > inGenious Targeting Laboratory (iTL) has acquired exclusive rights to a new gene modulating technology that allows various genetic outcomes to be produced from a single mouse model. In many experimental settings, the targeted inactivation of a gene in the form of a knockout mouse is the starting point for the generation of additional mouse strains with variant genetic alterations of the gene in question. Based on the tetracycline operon, the F.A.S.T.™ (Flexible Accelerated STOP TetO-knockin) system allows multiple mouse lines to be derived from a single targeted locus, comprising distinct genetic modalities such as: 1) a constitutive knockout; 2) its Cre-mediated tissue-specific rescue; 3) selective tTA-directed ectopic target gene expression within the knockout environment; 4) selective tTA-mediated target gene overexpression from within the wild-type environment; and 5) tTS-induced conditional knockout/knockdown of the target gene. Here, we summarize the versatility of multiple gain-and loss-of-function mouse lines of various genes that were produced using this integrative model approach. PDF

		An insight into the role of HNF4alpha in type 2 diabetes mellitus www.biobase-international.com > Hepatocyte nuclear factor 4 alpha is a transcriptional factor that acts in hepatocyte differentiation and glucose metabolism. HNF4alpha is a highly conserved member of the nuclear receptor family of ligand-dependent transcription factors. PDF

		A novel RNA detection reagent enables gene expression analysis and sorting of living cells www.millipore.com > Detecting gene expression has traditionally been limited to technologies that examine expression in lysed or fixed cell populations. The ability to detect RNAs in individual, live cells can enable an unequivocal assessment of gene expression changes that occur in direct response to specified perturbations. Determining which genes are up- or down-regulated in these perturbed cells provides insight into the relationships between gene expression networks and cell functions. To meet this challenge, we developed SmartFlare™ RNA Detection Probes, capable of detecting specific mRNAs and miRNAs in live, intact cells (Figure 1). This technology allows for carrier-free cellular endocytosis of the reagent, followed by detection and relative quantitative analysis of RNA levels. PDF

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