|Innovative technology that enables RNAi in difficult to transfect cells|
Christina Yamada, Kathryn Robinson, Allison St. Amand, Zaklina Strezoska, Greg Wardle, Anastasia Khvorova, Devin Leake
Investigations at Dharmacon have led to the development of innovative siRNA molecules that can be delivered into difficult-to-transfect cells without additional lipid reagents, virus, or instruments. This technology, Accell siRNA reagents, enables gene knockdown for functional genomic studies in a wide variety of cell types. In some instances, cells can be continuously dosed with Accell siRNAs to enable target gene knockdown for extended durations.
|LOHA Comprehensive Assay for Single Nucleotide Polymorphism, Copy Number Variants and Loss of Heterozygosity Using SureSelect Target Enrichment|
Kyeong Soo Jeong, Arjun Vadapalli, Ashutosh Ashutosh, Paula Costa, Brian Peter, Stephanie Fulmer-Smentek, Magnus Isaksson, Jayati Ghosh, Douglas Roberts, Holly Hogrefe
Here we describe a comprehensive assay that enables researchers to identify SNP, INDEL, CNV, and LOH using SureSelect target enrichment. This design can be employed as a standalone entity or in concert with other bait designs for SNP and INDEL detection. We also describe methods for data analysis and visualization.
|The Power Decoder simulator for the evaluation of pooled shRNA screen performance|
Jesse Stombaugh, Abel Licon, Žaklina Strezoska, Joshua Stahl, Sarah Bael Anderson, Michael Banos, Anja van Brabant Smith, Amanda Birmingham, Annaleen Vermeulen
Power Decoder (written in R and Python) simulates shRNA pooled screening experiments in silico to allow for the estimation of a screen’s statistical power. Populations of shRNAs were engineered in such a way that the magnitude of depletion and enrichment was known, then using the negative binomial distribution, an in silico model was developed to successfully resemble data from an actual laboratory experiment.
|Knockdown of long noncoding RNAs in breast cancer |
1 Jennii Luu, 2 Jesper Maag, 1 Yanny Handoko, 3 Richard Redvers, 3,4 Robin L. Anderson, 5 Maren M. Gross , 2 Marcel E. Dinger, and 1,3 Kaylene J. Simpson 1 Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre; 2 Genome Informatics, The Kinghorn Cancer Centre, The Garvan Institute of Medical Research; 3 Metastasis Research Laboratory, Peter MacCallum Cancer Centre, 4 Sir Peter MacCallum Department of Oncology, University of Melbourne;
RNAi global collaboration study using Lincode siRNA in a primary screen of tumor and nontumor breast cell lines. Hundreds of lncRNAs are found to affect viability and cell morphology of breast cancer. Presented at Keystone Symposia on Long Noncoding RNAs: From Evolution to Function, Mar 15 - Mar 20, 2015.
|Improved Small RNA Library Preparation Workflow for Next-Generation Sequencing|
Sabrina Shore, Jordana Henderson, Anton McCaffrey, Gerald Zon, Richard Hogrefe
We describe an optimized small RNA NGS library prep workflow using chemically modified adapters which suppresses adapter dimers, allows for RNA inputs down to 1 ng and eliminates the need for a gel purification step, thus allowing full automation not previously possible.
|Non-invasive Liquid Biopsy|
Frauke Henjes1, Lise Aagaard1, Darren Ellis1, Hannah Lindstroem1, Morten Oksvold2,3, Axl Neurauter1, Ketil W. Pedersen1.
Paramagnetic Dynabeads® magnetic beads provide a versatile, automation friendly tool for fast sensitive and high throughput isolation that can be tailored to isolate specific circulating biomarkers such as cells, exosomes and nucleic acids.
|Specificity of highly potent miRNA inhibitors|
Barbara Robertson, Andrew Dalby, Yuriy Fedorov, Jon Karpilow, Anastasia Khvorova1, Devin Leake, Annaleen Vermeulen
miRNA inhibitors are invaluable tools for elucidating the roles of miRNAs. However, potent inhibitors may also affect other miRNAs. To understand the potential cross-reactivity of miRNA inhibitors, various miRNA inhibitor designs were systematically tested. We demonstrate that mismatches both within and outside the seed region of the miRNA interfere with inhibition. Our findings indicate that features important for natural miRNA target recognition are also important for inhibitor specificity.
|Alternative miRNA design for therapeutic RNAi applications|
Anja van Brabant Smith, Barb Robertson, Annaleen Vermeulen, Christina Yamada, Angela Reynolds, Anastasia Khvorova, Devin Leake
For in vivo applications, the design of miRNA inhibitors and miRNA mimics must be optimized for stability and potency. However, stabilized miRNA mimic molecules can lose functionality compared to standard miRNA mimic molecules due, in part, to the activity of the stabilized passenger strand acting as a miRNA inhibitor. We discuss how mismatches affect the activity of the stabilized miRNA mimics, perhaps by generating a passenger strand that is less functional as an inhibitor molecule.
|Cas9 driven by an optimal promoter improves gene editing in eukaryotic cell lines when paired with synthetic crRNA and tracrRNA|
Amanda Haupt, Emily Anderson, Žaklina Strezoska, Hidevaldo Machado, Shawn McClelland, Maren Mayer, Adam Rocker, Annaleen Vermeulen, Amanda Birmingham, Melissa Kelley, Anja Smith
Presented here are results on the efficiency of using synthetic crRNA and tracrRNA to introduce gene editing events when co-transfected with a plasmid expressing Cas9. We explored the use of antibiotic and fluorescence activated cell sorting (FACS) methods for enrichment of cells that have undergone gene editing, and the use of multiple promoters to increase efficiency of gene editing with Cas9 and synthetic tracrRNA and crRNAs.
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