Nature Methods Application Notes e-UPDATE: 12 March 2013

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12 MARCH 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

		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

		Rapid and Non-Phenol Extraction of Circulating and Exosomal RNA from Bodily Fluids www.norgenbiotek.com > Norgen Biotek has developed various products (Plasma/Serum Circulating RNA Purification Kits and Exosome RNA Purification Kits) that are specifically optimized for purifying circulating or exosomal RNA (large RNA and microRNA) from plasma/serum or urine with the following advantages: Simple and Fast Procedure with no lengthy phenol:chloroform extraction, filtration or precipitation Scalable Purification of RNA from 50 µL to 5 mL of plasma/serum and 1 mL to 10 mL of urine High Yield, High Quality and Inhibitor-Free RNA isolated from all types of plasma, serum or urine 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

		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

		Understanding the impact of cancer cells' metabolic phenotype and intervention strategies www.seahorsebio.com > Cancer cells exhibit high rates of glycolysis, even in the presence of oxygen, a phenomenon known as the Warburg effect. While normal cells generate most of their ATP via oxidative phosphorylation, cancer cells are more reliant on glycolysis to generate ATP. Energy production through glycolysis results in a net production of protons; as glycolysis occurs, protons are extruded into the extracellular environment, acidifying the medium surrounding the cells or tissue. PDF

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