Open Biosystems guarantees that when you purchase a minimum of 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 controls and protocols and normalized for transfection efficiency. Appropriate transfection conditions should be confirmed using the appropriate positive controls provided in the kit 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 microRNAs 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.

Examples of validated GIPZ lentiviral shRNAmir targeting cancer genes GIPZ lentiviral shRNAmir directed against 6 different genes included in the validated cancer set. On average two out of three shRNAmir produced greater than 70% knockdown compared to the non-silencing control. Hatched line indicates 70% knockdown.

Consistent and stable knockdown of b-secretase (BACE) with 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:

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