EXPRESSION ARREST™ shRNAmir GENOME-WIDE LIBRARIES

MicroRNA-adapted shRNA (shRNAmir) for increased, specific and consistent knockdown.

UNIQUE microRNA-ADAPTED DESIGN

Advantages of shRNAmir design• Human microRNA-30 (mir-30) loop and context sequences adds

endogenous processing by Drosha

• Dicer recognition and specificity is increased by Drosha processing

• Dicer processing promotes active loading of the RISC complex

• Destabilizing 5’ end of antisense strand results in strand specific

incorporation into RISC

MicroRNA PROCESSING PATHWAY UTILIZED FOR shRNAmir

Developed in collaboration with Greg Hannon (CSHL) and Steve Elledge (Harvard) the shRNAmir libraries provide unique solutions for your diverse RNAi needs.

Features include:

• Unique microRNA-30 based design greatly

increases knockdown specificity and efficiency

• Pre-cloned into viral vectors

• Transient, stable and in vivo options for RNAi

• Transfection or infection options for delivery

• Guaranteed knockdown

Why Expression Arrest shRNAmir ?

shRNAmir design details 1,2,3

• Replaced mature microRNA sequence in primary mir-30 with gene-

specific shRNA duplexes

• Added Drosha cleavage site to the hairpin construct

shRNAmir produces more effective knockdown

Increased Drosha/Dicerprocessing

MoresiRNAs

Greaterknockdown= =

mir-30 context Droshacleavage site

mir-30 sequence mir-30 loop

Endogenous mir-30 primary transcript

Open biosystems mir-30 hairpin design

Drosha Dicer

Antisense sequence of shRNA

..U GAGC GXNN NNNNNNNNNNNNNNNNNNN UA-----

GUGAAGCCACAGAUGUA

NNNNNNNNNNNNNNNNNNNNNNUGCCUA..

miR5’ miR3’

BactR MammRPol II/III

LTR

sinLTR

shRNAmir

EXPRESSION ARREST™ shRNAmir SHOW 12-FOLD GREATER KNOCKDOWN

(A) shRNAmir constructs produce greater and more consis-

tent knockdown of target genes when compared against

conventionally designed shRNA to the same genes. Multiple

shRNAs against various proteasomal genes were tested in a

functional assay and the data averaged3.

(B) Northern blotting was used to detect the mature siRNA

produced after transfection of Hek293 cells with shRNA or

shRNAmir. Transfection was normalized using a co-delivered

dsRed expression plasmid. A 12-fold increase in the siRNA

produced after shRNAmir processing was detected relative

to non-mir shRNA processing3.

Evolution of RNAi technology

shRNAmir is superior to siRNA and conventional shRNA

Comparison of silencing triggers

siRNA, the earliest synthetic RNAi trigger

produces rapid, transient-only knockdown.

shRNA overcomes several limitations

inherent in siRNA, including the ability

to create stable knockdowns in vitro,

transduce hard to transfect cell lines, and

create animal models. shRNAmir does

all of the above and produces increased,

more specific knockdown relative to siRNA

or shRNA.

(A) (B)

shR

NA

mir

shR

NA

22mer

5S rRNA

MW

GUARANTEED KNOCKDOWN WITH EXPRESSION ARREST™ shRNAmir

All Expression Arrest shRNAmir constructs are guaranteed* to silence your target gene at least 70% when used along with shRNAmir controls and Arrest-In™ transfection reagent for shRNA delivery. These validated and opti-mized reagents come pre-packaged in RNAintro™ shRNA transfection kits and provide a controlled solution for gene silencing experiments.

RNAintro shRNA kits include:

• shRNAmir constructs of your choice—pick from human and mouse genomes

• Arrest-In™ Transfection Reagent for shRNA delivery

• Positive control—Luciferase or eGFP shRNA

• Negative control—Non-silencing shRNA construct

• Transfection efficiency control—ß gal reporter

shRNAmir TO KINESIN RELATED MOTOR PROTEIN eG5 LEADS TO DISRUPTION OF NORMAL CELL DIVI-SION

The kinesin related motor protein EG5 is known to be involved in centrosome separation. HEK293 cells were transfected with eG5 shRNA (v2hs_48561) and

48 hrs later stained for tubulin (anti-tubulin, green), DNA (DAPI, blue) and EG5 (anti-EG5, red). Targeting of the EG5 gene by shRNAmir results in the formation of

half spindles, and cells transfected with EG5 shRNAmir are arrested in mitosis and show monoastral microtubular arrays (see cell 1 remains in prometaphase).

By contrast, control cells (cell 2) show normal bipolar spindles and microtubule networks in mitosis. As a negative control, HEK293 cells were transfected with

a non-silencing shRNA.

References 1. Paddison, P et al (2004) Nature Methods 1:2, 163-167 2. Cleary, M et al (2004) Nature Methods 1:3, 241-248 3. Silva, JM et al (2005) Nature Genetics, in Press

OPEN BIOSYSTEMS EXPESSION ARREST™

Tubulin antibodyeG5 antibody

cell 1 cell 2

Save time with guaranteed to work shRNAmir

Triple overlay

do not have new image

STABLE KNOCKDOWN OF ß-SECRETASE (BACE) WITH shRNAmir

Five shRNAmir constructs directed against the human BACE gene

were transfected into SH-SY5Y neuroblastoma cells. Stable trans-

fected cells were selected with puromycin and assayed for BACE

protein five weeks later. BACE protein expression was reduced

between 85-99% by all five shRNAmir constructs tested. Actin was

used as a loading control.

Data courtesy of Dr Aleister Saunders and Preeti Khandelwal at

Drexel University

* When used with RNAintro protocols and normalized for transfection efficiency.

Anti BACE

Anti Actin

shRNAmir LIBRARY DELIVERABLES AND FORMATS

Querying for shRNA

1. Enter your gene symbol(s) or GenBank accession number(s) 2. Results prefaced by a yellow or green icon are exact matches to your search criteria 3. Click the View link for additional information including hairpin sequences 4. Order directly online. Constructs are shipped in 48 hours!

Make Open Biosystems your source for the latest RNAi technology

OPEN BIOSYSTEMS EXPESSION ARREST™

The Expression Arrest™ human and mouse shRNAmir libraries are available as individual constructs, rearrays of gene families and individually arrayed whole genome libraries.

Glycerol stocks and plasmid DNA formats of the shRNAmir libraries are available. Individual constructs are available as glycerol stocks while gene families and complete libraries are also available as transfection ready plasmid DNA.

OPEN BIOSYSTEMS EXPESSION ARREST™

GENOMICS E X P R E S S RNAi R E P R E S S PROTEOMICS D E T E C T

Full-length cDNAsHuman ORFsSNAP-tag™ protein labeling kits

MicroRNA-adapted shRNA librariesRNAintro shRNA transfection kits Drosophila and C. elegans RNAi libraries

Custom antibodiesCustom peptidesCatalog antibodies

Your expert partner for Genomic, RNAi, and Proteomic resources.

*SNAP-tag is a trademark of Covalys Biosciences AG

Go to www.openbiosystems.com to place an order or call: 1-888-412-2225 to speak with a representative.

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