Thermo Scientific Dharmacon Accell sirna A new world of RNAi discovery

Transcription

1 Thermo Scientific Dharmacon sirna A new world of RNAi discovery Delivery to any cell type without transfection reagents or viral vectors Extraordinary ease-of-use for rapid results Expert designs and modifications for specific target silencing

2 sirna delivery into any cell type empowers RNAi innovation RNAi research no longer is constrained by cell types that are resistant to conventional lipid-based delivery reagents. Thermo Scientific Dharmacon sirna provides delivery into any cell type for unprecedented experimental flexibility and discovery. Effective delivery with no transfection reagent or viral vector required Novel sirna modifications for uptake, stability, and specificity Proven performance in primary, differentiated, and other difficult-to-transfect cell types delivery into any cell type Efficient delivery of Dharmacon Red sirna to SH-SY5Y and MCF-7 cells Dharmacon sirna provides reproducible, reliable results across cell lines Uptake of sirna confirmed by fluorescence microscopy. SH-SY5Y (top panel) and MCF-7 cells (lower panel) were treated with Red Cyclophilin B Control sirna in delivery media. (Red fluorescence = Cytoplasmic localization of sirna; Blue = Nuclear Hoechst dye). HEK293 FKHR-U2OS HeLa S3 LAN-5 HuVEC MCF-a SH-SY5Y SKOV3 THP- MK2-U2OS HuH-7 T Cells Jurkat IMR-32 mes-d3 mc2c2 mprihepat m3t3l Control Dharmacon sirna has delivered effective knockdown in every cell type tested to date Human Suspension Cells Human Differentiated Stem Cells Human, Mouse, and Rat Primary Cells Other Mouse and Rat Cells Human Adherent Cells Jurkat THP- IM-9 Osteoblasts and adipocytes derived from hmsc HUVEC HUASMC hmsc PBMC Primary human astrocytes Primary human T cells Primary human keratinocytes Primary mouse hepatocytes Primary rat cortical neurons Primary rat striatum neurons 3T3 NIH ES-D3 3T3 L Rat2 C2C2 H9c2 PC-2 SH-SY5Y IMR32 LAN5 A-375 HeLa HeLa S3 LNCap MCF-A 293T MCF-7 SK-BR3 OVCAR-3 Huh7 GTM-3 DU45 HT8 DLD- HepG2 U2OS HEK293 U87MG MIA PaCa-2 NCI/ADR-RES

3 Extraordinary ease-of-use for rapid, high confidence results Dharmacon sirna reagents are specially modified for uptake by any cell type without a separate delivery reagent - no complexing wait time, no wash steps. Simply combine Dharmacon sirna with optimized sirna delivery media* and add directly to cells. Achieve faster time-to-results with a two-step delivery protocol Enjoy long-term reproducibility with delivery mix stability of up to 9 months at 4ºC Simplify RNAi delivery conditions with a single, validated delivery media formulation* ease-of-use A simple delivery protocol with Dharmacon sirna makes targeted gene knockdown effortless sirna reagents deliver effective knockdown without cytotoxicity. Eighteen cell lines were treated with Cyclophilin B sirna in delivery media and assayed for knockdown at 72 hours. mrna expression was determined by QuantiGene branched DNA assay (Panomics) and cell viability was determined by alamarblue (Biosource International). sirna (stock solution) delivery mix ( sirna) 2 sirna delivery media * sirna should always be used with delivery media (Cat# B-5). This is a serumfree media specially formulated and optimized for use with sirna. It has undergone thorough testing and successful validation in over 4 cell lines A B C D E F G H 2 Combine sirna and delivery media Add delivery mix directly to cells and incubate 72 hours

4 Accelerate time to results and leave lengthy delivery optimization behind Dharmacon sirna reagents eliminate the need for complicated delivery optimization by consistently providing effective sirna delivery and knockdown at an unprecedented range of experimental conditions. In addition to simplifying single-gene silencing experiments, Dharmacon sirna lends itself to high-confidence screening with higher reproducibility. Save time and money by eliminating transfection reagents Reduce the need for cell type-specific delivery protocols Expand assay possibilities with more cell types and broader RNAi conditions accelerate time to results Narrow windows of optimization hinder reproducibility. MK2-U2OS (Thermo Scientific BioImage) cells were transfected with nm sigenome Cyclophilin B Control sirna with three concentrations of Thermo Scientific DharmaFECT Transfection Reagent across three cell densities and assayed at 24 hours post-transfection. Dharmacon sirna application is superior to lipid transfection at a wide range of conditions Lipid optimization.5 µl µl. µl µl.2 µl µl Untreatedted.5 5 µl µl. µl µl.2 2 µl µl Untreated ted.5 5 µl µl 5K K 2K Dharmacon application. µl µl.22 µl µl Untreatedted Boxed areas indicate optimal conditions - cellular viability greater than 75% and target gene knockdown greater than 75%. Broad windows of optimization provide experimental flexibility and high confidence results. MK2-U2OS cells were treated with.,.5, and Cyclophiln B Control sirna across three cell densities and assayed at 72 hours post-transfection Cyclophilin B expression was determined by QuantiGene branched DNA assay (Panomics) and cell viability was determined by alamarblue (Biosource International)..5. Untreated ted Untreatedted 5K K 2K Untreated Untreatedted

5 Novel delivery causes minimal cellular perturbation for unmatched clarity of results Imagine your results free from the toxicity and inflammatory response typically associated with conventional lipid transfection reagents. The passive nature of Dharmacon sirna uptake avoids adverse cellular responses that could interfere with analysis of target gene knockdown. This new paradigm for sirna delivery offers: No detectable inflammatory response Sustained viability at a range of cellular densities Elimination of non-specific lipid effects for pure silencing results clarity of results Reduce off-targets and delivery effects with Dharmacon sirna --X 4X Scale: Scale + 4X +X 5 DBI PPIB LMNA 3 HIFA RB RHOA RAC CDC A RN cce A ll #4 A de cc l e m iver ll ed y ia si si A RN cce A ll #3 si Ac RN ce A ll #2 si Ac RN ce A ll # on e 2 al pi d sirna μm working concentration induces little to no off-target effects. Total RNA from HeLa cells treated with SMARTpool reagents targeting different genes was measured by microarray analysis (Agilent Whole Genome Array, 4 x 44K format). The number of offtargets (showing 2-fold or more downregulation) are indicated. CDK4 Li # of Off-target Genes MET IL-8 ProteinExpression (pg/ml) Target Knockdown Dharmacon sirna delivery does not induce expression of the IL-8 inflammatory protein HeLa S3 cells were treated with either lipid alone (DharmaFECT transfection reagent), sirna (either #, 2, 3 or 4) targeting diazepam binding inhibitor or delivery media alone. 72 hours after transfection, the cellular supernatant was analyzed for the cytokine IL-8 and eight other interferon response genes (IL-α, IL-β, IL-2, IL-6, IL-, IL-2p7, IFNγ, TNFα) using the SearchLight array platform.

6 Dharmacon sirna delivers a proven silencing strategy Dharmacon sirna provides specific knockdown of your target gene by combining SMARTpool technology - recognized to enhance overall functionality and specificity - with additional proven strategies: Validated SMARTselection sirna design algorithm for effective silencing Seed region filters for toxic and mirna-like motifs along with seed frequency analysis to eliminate designs with high likelihood of off-target silencing Specificity-enhancing modifications on both strands to reduce off-target effects proven silencing strategy Dharmacon sirna reagents successfully silence target genes across cell types 2 Achieve effective target knockdown across cell types. HeLa S3, Rat-2, 3T3L, Jurkat, THP-, NHA, and SH-SY5Y cells were treated with μm SMARTpool sirna targeting various genes or Non-targeting control (NTC) and assayed for knockdown at 72 hours mrna expression was determined by QuantiGene branched DNA assay (Panomics) and cell viability was determined by alamarblue (Biosource International). LMNA CDC2 TP53 AKT RB HRI MET DBI rppib rgapdh mgapdh mppib PTPN RhoA Csk RhoA PPIB RhoA PPIB CDC2 TP53 AKT RB HRI DBI RAC RhoA SRC NTC Untreated HeLa S3 Rat-2 3T3 L Jurkat THP- NHA SH-SY5Y Controls egfp expression silenced by Dharmacon sirna FKHR-U2OS cells (BioImage) were treated with μm egfp Control sirna or Non-targeting Control sirna # in delivery media. Reduction of egfp expression is visualized using Cellomics ArrayScan V TI (green= GFP expression; blue= nuclear Hoechst dye). Delivery Media Only egfp Control sirna Non-targeting Control sirna #

7 New delivery technology. New possibilities. A new world of RNAi is waiting. The passive nature of Dharmacon sirna uptake lends itself to gene silencing applications not possible with other delivery techniques: Repeated dosing for extended duration knockdown - useful for study of long-lived proteins and downstream cellular responses Transient silencing of multiple genes with multiplex delivery Combinatorial approaches to silence in cells stably transduced using SMARTvector shrna technology new possibilities Demonstration of extended duration silencing with continuous dosing of Dharmacon sirna HeLa and SH-SY5Y cells were treated with Cyclophilin B Control sirna at multiple intervals for sustained target knockdown. HeLa cells showed sustained knockdown for 3 days (9 passages), SH-SY5Y for 2 days (5 passages). At each passage, cells were split and cultured in the delivery mix. Target knockdown was reassessed at 24 (HeLa) or 48 hr (SH-SY5Y) intervals following each sirna treatment. Day 3 Day 7 Day Day 5 Day 9 Day 3 Day 7 Day Day 5 Day 9 Day 2 Day 6 Day Day 4 Day 8 Day 22 Day 26 Day 3 Day 2 Day 6 Day Day 4 Day 8 Day 22 Day 26 Day 3 PPIB sirna SH-SY5Y NTC sirna PPIB sirna HeLa NTC sirna mrna expression was determined by QuantiGene bdna assay (Panomics) and cell viability was determined by alamarblue (Biosource International, Inc). Get started at Predesigned Dharmacon sirna reagents Dharmacon sirna delivery media Dharmacon RNAi controls Dharmacon sirna libraries Simply search for your human, mouse, or rat gene and click to order Optimized for use with Dharmacon sirna to virtually eliminate delivery optimization Validated positive controls for Cyclophilin B, GAPD, and egfp, and non-targeting negative controls for high confidence results Check our website for a growing list of available gene collections

8 Thermo Scientific Dharmacon sirna Contact Information North America 265 Crescent Dr., Suite Lafayette, CO 826 Toll free: Tel: Fax: Belgium & distributors Industriezone III Industrielaan Erembodegem Tel: Fax: France BP Brebières Tel: Fax: The Netherlands Postbus AA Etten-Leur Tel: Fax: Germany Adenauerallee Bonn Tel: Fax: United Kingdom Unit 9 Atley Way, North Nelson Industrial Estate Cramlington, Northumberland NE23 WA Tel: Fax: Switzerland Place Pépinet 2 Lausanne Tel: Fax: US web: Euro web: US dharmacon.lab@thermofisher.com Euro perbio.eurotech@thermofisher.com. 28 Thermo Fisher Scientific Inc. All rights reserved. QuantiGene is a trademark of Bayer. alamarblue is a trademark of Accumed International, Inc. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. US Literature # 66-7-K--U Euro Literature # PB 27 75