Guaranteed knockdown with shRNAmir

Open Biosystems guarantees that when you purchase upto three shRNAmir to the same target, at least one of the shRNAmir constructs will reduce target mRNA levels by 70% or more when used with RNAintro starter kit protocols and normalized for transfection efficiency. Appropriate transfection conditions should be confirmed using the luciferase or eGFP positive controls and the % knockdown should be compared to cells transfected with the non-silencing shRNAmir. All transfections should be carried out using Arrest-In transfection reagent supplied in the RNAintro kits.

shRNAmir Library Validation
The shRNAmir libraries are a new generation of shRNA libraries that takes into consideration advances in the understanding of microRNA biogenesis. In these constructs the shRNA is harbored within the backbone of the primary microRNA transcript. This natural configuration proved to be 12 times more efficient in the production of the mature synthetic miRNAs that the first generation design. Additionally as detailed in Silva et al (2005) biochemical characterization of processing of these synthetic micrRNAs allow prediction of the mature small RNA proucts generated from each vector. This has allowed selection of target sequences that maximize efficiency by directing preferential incorporation of the correct strand into RISC. The human and mouse shRNAmir libraries have been produced using these criteria. A subset of shRNAs from these libraries have been assayed for their ability to knockdown the expression of targeted genes by quantitative RT-PCR and also in a phenotypic (functional) assay. See graph below:

shRNAmir to Kinesin related motor protein EG5 leads to disruption of cell division


Figure 2: 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 shRNA results in the formation of half spindles, and transfected cells are arrested in mitosis and show monoastral microtubular arrays (cell 1 reamains 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 non-silencing shRNAmir.

Figure 3: Five shRNAmir constructs were directed against human b-secretase gene were transfected into SH-SY5Y neuroblastoma cells. Cells were selected with puromycin and assayed for BACE protein expression 5 weeks later. Normalized BACE protein expression is greatly reduced (85-99%) by all five shRNAmir constructs tested.
Data courtesy of Dr Aleister Saunders and Preeti Khandelwal at Drexel University.

Further details on the shRNAmir library construction and validation in vitro and in vivo can be obtained from the following publications:

Paddison, P et al (2004) "Cloning of short hairpin RNAs for gene knockdown in mammalian cells" Nature Methods Vol1:2, 163-167.
Cleary, M et al (2004) "Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis" Nature Methods Vol 1:3, 241-248.
Dickens RA et al (2005) "Probing tumor phenotypes using stable and regulated synthetic microRNA precursors" Nature Genetics Vol 37:11, 1289.
Silva J et al (2005) "Second-generation shRNA libraries covering the mouse and human genomes" Nature Genetics, Vol 37:11, 1281-88.
Westbrook et al (2005) "A genetic screen for candidate tumor suppressors identifies REST" Cell, Vol. 121, 837–848.


Figure1: 36 shRNAmir directed against proteasomal genes were tested for their ability to suppress mRNA expression (lower panel) QRTPCRs were performed 24 hrs after transfection of Hela cells at an average efficiency of 80% as measured by the co-transfected normailization reporter (dsRED). For comparison functional assays for proteasome inhibition (upper panel) were performed in parallel (From Silva et al 2005)

Guaranteed knockdown with shRNAmir

Send mail to webmaster with questions or comments about this web site. Copyright © 2005 Gentaur BVBA Last modified: feb-07

Send mail to webmaster with questions or comments about this web site.
Copyright © 2005 Gentaur BVBA
Last modified: feb-07