RNA Interference with Guaranteed Knockdown!

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1 RNA Iterferece with Guarateed Kockdow! microrna, shrna, & sirna microrna detectio with SYBR-Gree qpcr experimets microrna expressio microrna target validatio with 3'-UTR cloes shrna & sirna mediated kockdow

2 microrna Expressio Plasmids Comprehesive coverage for huma ad mouse OriGee provides cloes for over-expressio of microrna of your choice. OriGee s microrna precursor cotais pre-micorna (60-70t) with ts up- ad dow-stream of flakig sequece. It was amplified from huma geomic DNA ad cloed ito OriGee s pcmv6-mir Vector. Upo trasfectio, the cellular machiery will process the CMV-drive expressio of mirna precursor ito mature mirna ad cellular fuctio ca be aalyzed. Pri-miRNA Geome wide mirna coverage 652 huma ad 486 mouse Sequece cofirmatio of the precursor microrna mgppp Drosha AAAAAA GFP for trasfectio moitorig Drosha cut Neomyci selectio for stable cell establishmet GFP trasfectio of microrna expressio plasmids i HEK293 cells Pre-miRNA 5 3 Dicer cut Dicer Mir205 Mir143 Mir34b Mature mirna 5 3 Empty Vector No-trasfected MICRORNA EXPRESSION PLASMIDS Sold idividually as 10ug trasfectio-ready DNA or ca be purchased as followig sets RISC complex Catalog No. SC SC SC SC Descriptio Mouse mirna expressio plasmid set (486 vectors, 10ug each i 2-D bar coded tubes) Huma mirna expressio plasmid set (652 vectors, 10ug each i 2-D bar coded tubes) Mouse mirna expressio plasmid set (486 vectors, 2ug each i 96-well plates) Huma mirna expressio plasmid set (652 vectors, 2ug each i 96-well plates)

3 3'-UTR Reporter Cloes for mirna Target Validatio Luciferase reporter assays for huma geome The 3'-UTR plasmids provide quatitative assessmet of the ihibitory effect betwee mirnas ad their potetial target gees. The 3'-UTR sequece of a gee was cloed dowstream of the firefly luciferase gee. The chimeric trascript level is regulated by its iteractio with mirna(s), which results i varied luciferase activity quatifiable by a colorometric assay. MicroRNAs ad their 3'-UTR targets ca be assayed for edogeous microrna activity or for overexpressed microrna i a co-trasfectio experimet. SV40 P STOP STOP NEO IRES Luciferase 3'-UTR Target SV40 P STOP STOP NEO IRES Luciferase INTERACTION IRES-Luc Traslatio microrna RISC complex NO INTERACTION IRES-Luc Traslatio microrna RISC complex 3'-UTR Target AAAAAA 3'-UTR Target AAAAAA LOW LEVEL LUC TRANSLATION Luciferase LUC TRANSLATION Luciferase Iteractio betwee mirna ad UTR. Reduced luciferase expressio No iteractio betwee mirna ad UTR. No effect o luciferase expressio Geome wide coverage >20,000 huma gee Firefly luciferase as the easy-to-assay reporter RFP for trasfectio moitorig High sesitivity from IRES-drive traslatio of the expressio cassette SV40 F1 Ori CMV RFP Neo r / Ka r IRES pmirtarget Vector 7.9 kb PolyA ColE1 Luciferase PolyA EcoR I Sgf I Asc I Rsr II Miu I Not I Xba I pcmv - Mir + Mir205 target pcmv - Mir205 + Mir205 target OriGee has used a ew desig adapted from C.P.Peterse et al. 2006, to dramatically icrease the sesitivity of detectio by decreasig the 3'UTRluciferase reporter expressio to a very low level.

4 qstar microrna qpcr Detectio Assays Quatify your results dow to the absolute copy umber! OriGee s uique primer-based, SYBR Gree qpcr microrna detectio system ot oly offers researchers a fast ad simple method for profilig mirna expressio levels, but also provides meas to quatify the results dow to the absolute copy umber of mirna. Step I: PolyA Tailig mirna Step II: cdna Sythesis PolyA Polymerase AAAAAAAAAAAAAAAA 3' Geome wide coverage of huma ad mouse mirna Determie absolute copy umber of your mirna with template stadards Detect mirna directly from total RNA samples AAAAAAAAAAAAAAAA 3' GTTTTTTTTTTTTTTTTTTTTCGCACTTAGTCTTCAGAGTCTATGCGTCTGTACAAG 5' A C Achor Oligo dt Adaptors Step III: qpcr with mirna Specific Primer MMLV Reverse Trascriptase AAAAAAAAAAAAAAAA GTTTTTTTTTTTTTTTTTTTTCGCACTTAGTCTTCAGAGTCTATGCGTCTGTACAAG 5' A C mirna specific primer GTTTTTTTTTTTTTTTTTTTTCGCACTTAGTCTTCAGAGTCTATGCGTCTGTACAAG 5' 3'CTCTATGCGTCTGTACAAG 5' Uiversal primer PRODUCTS OFFERED IN qpcr microrna DETECTION SYSTEM 1 Total RNA Purificatio Kit 2 First Strad cdna Sythesis Kit 3 mirna Primer Pairs or mirna Primer Paels 4 mirna Template Stadards 5 SYBR Gree Master Mix *Compoets 2,3 & 4 are uique ad should oly be used alogside OriGee s qpcr mirna detectio system First-strad cdna Sythesis Kit Two-step protocol kit Additio of poly (A) tail to RNA sample Use of achor liker oligo dt to sythesize first-strad cdna microrna Primers & Paels Offered as idividual primers, geome wide paels, & custom-mixed paels Pre-validated i a experimet agaist ormal ad ovaria cacer samples microrna Copy Number Stadards Uique offerig oly from OriGee Determie the absolute trascript copy umber of a experimet sample usig the stadard curve method

5 Dicer-Substrate Techology for shrna & sirna 27mer-29mer deliverig higher kockdow tha traditioal 21mer By its optimal legth, HuSH-29 shrna & Trilecer-27 sirna have the advatages of improved efficacy ad miimal iterfero respose. The legth ad desig of OriGee s RNAi substrates are importat improvemets over the use of traditioal 21mer desigs. Loger shrna & sirna costructs appear to eter the RNAi pathway more efficietly ad result i much higher potecy ad specificity tha shorter RNAi forms. However, i most mammalia cells, log double-straded RNA provokes a iterfero respose as part of a ativiral defese. To overcome this obstacle, OriGee desigs shrnas & sirna of less tha 30 base pairs i legth, which evade he mammalia immue system while still iitiatig strog ad specific gee silecig. A comparative study of differet sirna desigs was coducted i a Nature Publicatio (Referece 1). Accordig to the publicatio, short RNAs that are log eough to serve as Dicer substrates (D-siRNA) ca ofte evoke more potet RNA iterferece tha the correspodig 21-t sirnas; this is probably a cosequece of the physical hadoff of the Dicerproduced sirnas to the RNA-iduced silecig complex. HOW DICER-SUBSTRATE RNAi WORKS Dicer sirna-27 or shrna-29 Duplex 21 mer sirna or shrna IT NF positive cells (et % from LPS vs, liposome oly) t sirna TNF 21-t sitnf Dissociate Dicer RISC M 0.2 M 1.0 M 5.0 M 25.0 M Compariso of gee kockdow usig dicer-substrate sirna ad 21 mer sirna KEY PUBLICATIONS ON DICER-SUBSTRATE TECHNOLOGY 1. Ratioal desig ad i vitro ad i vivo delivery of Dicer substrate sirna, Nature Protocols 1, (2006) 2. Priciples of Dicer Substrate (D-siRNA) Desig ad Fuctio, Methods i Molecular Biology, 442: Cap RISC Target mrna AAAAA Dicer mrna Degradatio

6 HuSH-29 shrna Guarateed Gee Kockdow Comprehesive coverage of huma, mouse & rat gees DOUBLE KNOCKDOWN EXPERIMENT 29mer shrna: higher potecy, miimal iterfero respose Four retroviral vectors, icludig GFP & RFP for trasfectio moitorig ad multiple selectio markers to carry out double kockdow experimets Trasiet or stable trasfectio or retroviral ifectio shrna kit: 4 gee-specific costructs + 2 egative cotrols Scrambled shrna (GFP Vector) + Scrambled shrna (RFP Vector) shrna agaist RFP (GFP Vector) + shrna agaist GFP (RFP Vector) SCHEMATIC DIAGRAM OF GFP & RFP VECTORS Both vectors have bee validated through publicatios. Loop Target Sequece A A G C A Target Sequece RC* NNNNNNNNNNNNNNNNNNNNNNNNNNNNN T G NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN TTTTTTG GAA 29t 29t Loop Target Sequece A A G C A Target Sequece RC* NNNNNNNNNNNNNNNNNNNNNNNNNNNNN T G NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN TTTTTTG GAA 29t 29t U6 Pr SV40 Pr U6 Pr SV40 Pr 5 LTR Puro r 5 LTR Puro r Ka r pgfp-v-rs (7584 bp) 3 LTR CAM r prfp-c-rs (7452 bp) 3 LTR CMV Pr tgfp MCS pbs Ori RC*: reverse complemet CMV Pr trfp MCS pbs Ori RC*: reverse complemet Exact-shRNA Service Desig ay shrna or mirna with custom sythesis service The same superior desig that is available for our pre-desiged HuSH-29 sets is also offered through our Exact-shRNA service. Self-desig or let OriGee desig for you Target species other tha huma, mouse or rat Itegrate a effective sirna sequece ito a shrna vector Reproduce the result of a published shrna sequece

7 I vitro assessmet of shrna targetig HIF1A Dowregulatio of HIF1A Expressio by HuSH Costructs I vivo effects of shrna targetig Sprouty4 (Cat# TR509780) PLoS ONE. 2009; 4(5): e5467 Cotrol shrna Cotrol prs Vector TI HIF1A HuSH Vectors i HuSH TR Kit TI TI TI o-effective shgfp HIF1A Blot: Ati-HIF1A Spry2/Spry4 shrna WT Spry 4 KO Erk1 Erk2 ReBlot: Ati-ERK1/2 KEY PUBLICATIONS 1. Suppressio of Sproutys Has a Therapeutic Effect for a Mouse Model of Ischemia by Ehacig Agiogeesis, PLoS ONE. 2009; 4(5): e5467 [I vivo applicatio] 2. Adroge-iduced TOP2B-mediated double-strad breaks ad prostate cacer gee rearragemets, Nature Geetics (4 July 2010) doi: /g.613 Article [shrna targetig TOP2B i GFP vector] 3. EMX2 is epigeetically sileced ad suppresses growth i huma lug cacer, Ocogee (9 August 2010) doi: /oc Short Commuicatio [shrna targetig EMX2 i RFP Vector] Trilecer-27 sirna Guarateed gee silecig for huma gees Similar to HuSH-29 shrna, OriGee s Trilecer-27 sirna utilizes a 27mer Dicer-Substrate desig that has the advatages over traditioal 21mer of improved efficacy ad miimal iterfero respose. Geomewide coverage agaist huma Higher potecy & miimal iterfero respose sirna kit: 3 gee-specific sirnas + 1 egative cotrol sitran 1.0 sirna TRANSFECTION REAGENT Dual purpose reaget trasfect both sirna duplex ad correspodig cdna cloe High trasfectio efficiecy ad low cytotoxicity

8 OriGee, Your Parter i Gee Research ad Beyod KEY TECHNOLOGIES AND PRODUCTS Full-legth cdna cloes, ORF cloes i expressio-ready vectors Gee sythesis: ay gee, ay variat, ay vector RNAi research reagets: shrna, sirna, ad mirna fuctio ad detectio SYBR Gree qpcr assays for mrna ad mirna detectio, primer paels Recombiat huma proteis ad over-expressio lysates TrueMAB moocloal atibodies Lumiex multiplex immuoassays Cacer tissue biorepository, TMA, RPPA, ad TissueSca qpcr arrays UltraMAB validated IHC atibodies OriGee USA 9620 Medical Ceter Drive, Suite 200 Rockville, MD OriGee Gee Sythesis Blue Hero Biotech th Aveue SE #100 Bothell, WA USA OriGee CHINA HuiLogSe Iteratioal Sciece & Techology Idustry Park Build 11, No.99, Kechuag Fourteeth Street, D4 Beijig, CHINA