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Unique microRNA adapted design

shRNAmir produce 12 fold greater processed siRNA

Northern blotting was used to detect the mature siRNA produced after transfection of Hek 293 cells with shRNA or shRNAmir. 12 fold greater siRNA was detected after shRNAmir processing vs shRNA processing (from Silva et al 2005)

shRNAmir design
Expression Arrest™ shRNAmir triggers have been designed to mimic a natural microRNA primary transcript and each target sequence has been selected based on thermodynamic criteria for optimal small RNA performance. Validation of this design is detailed in Silva et al (2005) showing a substantial increase in knockdown efficiency.

Advantages of shRNAmir design
  • Replaced mature microRNA sequence in mir-30 with gene specific duplexes
  • Adding mir-30 loop and context sequences adds endogenous processing by Drosha which increases subsequent Dicer recognition and specificity
  • Dicer processing promotes active loading into the RISC complex
  • Rules based design include destabilizing the 5"end of the antisense strand for strand specific incorporation into RISC
Increased Drosha/Dicer processing=More siRNA=Greater knockdown

 

shRNAmir produce increased knockdown


shRNAmir constructs produce greater and more consistent knockdown of target genes when compared against conventionally designed shRNA to the same genes. Multiple shRNAs againstvarious proteasomal genes were tested in a functional assay and the data averaged.


 
References:
  1. Paddison, P et al (2004) 'Cloning of short hairpin RNAs for gene knockdown in mammalian cells' Nature Methods Vol 1:2, 163-167
  2. Silva, J et al (2005) 'Second-generation shRNA libraries covering the human and mouse genomes' Nature Genetics Vol 37:11, 1281-88.
  3. Lee, Y et al (2002) ‘MicroRNA maturation: stepwise processing and subcellular localization' EMBO Vol 21, 17: 4663-4670.
  4. Chendrimada et al (2005) 'TBRP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing' Nature Vol 436, 740-744.
  5. Gregory et al (2005) 'Human RISC couples microRNA biogenesis and post-transcriptional gene silencing' Cell, Vol 123, 631-640.
 

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