platinum select tm shrna-mir - transomic 1. harnessing the endogenous microrna pathway to trigger...
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PLATINUM SelectTM shRNA-mir Incorporating advances in shRNA design for enhanced
potency and specificity
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Figure 1. Harnessing the endogenous microRNA pathway to trigger RNAi.
Green cartoons (on the right) show where siRNA, shRNA and shRNA-mir enter
the endogenous microRNA pathway to trigger RNAi.
PLATINUM SelectshRNA-mir
Enhanced potency for 100% guaranteed* knockdown
microRNA-30 context for enhanced speci!city and reduced toxicity
shRNA-mir designs are scored and ranked per gene
Vector expressing shRNA and
selection markers
Viral promoter - e!cient shRNA-mir expression
Puromycin resistance - select stable integrants for long-term knockdown
TurboGFP - marker of shRNA-mir expression
shERWOOD Algorithm, microRNA
context for 100% guaranteed potency
PLATINUM Select MLP Retroviral microRNA-adapted
short hairpin RNA (shRNA-mir) are designed using the
proprietary shERWOOD algorithm developed in Dr. Greg
Hannon’s laboratory at Cold Spring Harbor Laboratory
(CSHL). Advantages include:
shRNA-mir have a proven advantage
over simple stem loop shRNA
shRNA-mir constructs embed the silencing sequence in a
primary microRNA transcript resulting in increased speci-
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compared directly against simple stem loop shRNA. Simple
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(Beer et al 2010, Castanatto et al 2007, Pan et al 2011).
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WR[LFLW\ (McBride et al 2008).
A high-throughput “sensor” assay was used by the Hannon
lab to test 270,000 shRNA-mir sequences for their ability to
knock down their target (or sensor) gene fused to a !uo-
rescent reporter “Venus”. shRNAs that e"ectively inhibited
the expression of their gene targets in the sensor would also
inhibit expression of the reporter gene, resulting in loss of
!uorescence (Figure 2). shRNA sequences targeting every
gene in the human genome were tested for potency using
the sensor assay and the data on sequence requirements for
the rare, potent hairpins were used to train the shERWOOD
algorithm (unpublished data).
Sensor scored hairpins were bench tested to verify the correlation to knockdown potency of the high and low scoring
shRNA sequences. Sensor scores correlated very well with knockdown potency. Western blot data show that the highest
scoring haipins produced almost complete knockdown of protein expression at single copy. In contrast the low scoring
hairpins did not perform well.
shERWOOD Algorithm trained using functional data from the Sensor Assay
Sensor scores correlate with shRNA potency
Figure 3. Western blots showing knockdown data for two sensor scored genes Trp53 and Bcl2. Protein knockdown was quantitated after transduction at single copy (MOI=0.3) of both good and bad scoring hairpins. As shown, sensor scores
correlated with knockdown potency. The highest scoring hairpins produced almost complete knockdown of protein expression at single copy. In contrast, the low scoring haipins did not perform well. Adapted from Fellman et al 2001.
Figure 2. Schematic showing the sensor assay used to test 270,000 sequences and
train the shERWOOD algorithm.
Enabling Discovery Across the Genome
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Trp53
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tiled across the nine genes (Fellman et al. 2011���7R�GHWHUPLQH�WKH�OLNHO\�VHQVRU�VFRUHV�RI�KDLUSLQV�FRQWDLQHG�LQ�WKH�QHZ�
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.
shERWOOD Algorithm predictions score well in the sensor assay.
Figure 4A. shERWOOD algorithm predictions corelate with sensor scores (corr=0.73). 4B. Red bars show data extracted from 4A to indicate the distribution of all 20,000 hairpins for 9 genes. Note the large proportion of poor hairpins and small proportion of potent hairpins (red bars). Blue bars correspond to the top 4 ranked shERWOOD predictions in the Platinum Select shRNA-mir library. Nine genes - Bcl2, Mcl1, Hras, Kras, Myc, PCNA, Rpa3, Trp53, hMyc.
shERWOOD predicted ranks correlate with potent knockdown
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the empty vector control.
Figure 5. Western blot showing protein knockdown produced by several shERWOOD predicted hairpins targeting PTEN after transduction at single copy (MOI=0.3) in HEK293T cells. Cells were puromycin selected and western blots performed using the Santa Cruz PTEN antibody. Boxed hairpins are the top 4 ranked hairpins targeting PTEN in the Platinum Select MLP retroviral shRNA-mir library.
shER
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scor
es
KD%
2
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2
0
-2
-4
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-8
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shER
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Pre
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Freq
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- 8 - 6 - 4 - 2 0 2 4 Sensor Score
- 8 - 6 - 4 - 2 0 2 4 Sensor Score
Use the Fetch my gene™ search tool to #nd shRNA-mir clones targeting your gene of interest. Fetch my gene™ is designed to help
you easily #nd products for your gene of interest and con#rm your results using the gene information provided.
3. Con!rm gene and product
4. Add to cart
Input your search terminto Fetch my geneTM.
1. Search
Quickly !lter producttype using tabs.
2. Choose product type
Clone speci!c information provided through link to clone
information page.
Validate your gene ofinterest with additional
gene information provided.
Click add to cart to purchase or continue
shopping.
100% guaranteed knockdown
All shRNA-mir constructs in a target gene set or starter kit are guaranteed to knock down mRNA expression >70%. Cell line of choice
should show target gene expression and demonstrate gene knockdown using positive
and negative controls.
ReferencesFellman et al., 2011. Mol Cell. 18; 41(6):733-7.Castanotto et al., 2007. Nucleic Acids Res. 35(15):5154-51.McBride et al., 2008. PNAS 105; 15, 5868-5873.Beer et al., 2010. Mol Ther 18(1):161-170.Pan et al., 2011. FEBS Lett. 6;585(7):1025-1030.
The PLATINUM Select MLP Retroviral shRNA-mir collections targeting human and mouse genomes are available as individual
shRNA-mir constructs, target gene sets, starter kits, gene families and pathway sets as well as genome libraries.
Custom rearrays are also available. Contact [email protected] for more information
or to submit your gene list.
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