How is the shRNA designed for the pKD mammalian siRNA expression plasmid?
In selecting the nucleotides for the shRNA, we follow the basic principle established by Elbashir S.M., …and Tuschl T. (Methods 26 (2002) 199-213). And in forming the shRNA design, we follow the basic procedure reported by Brummelkamp TR, …Agami R. (Sciencexpress, 21 March 2002, 1-6). The siRNA sequence specific to the target is 19 nucleotides in length. This sequence is repeated in the DNA oligo as a reverse compliment. The two sequences are separated by a 9 nt random sequence that will serve as the “loop” in the short hairpin RNA (shRNA). The DNA oligo contains the 5dT terminator sequence as the most 3’ element upstream of the restriction site for cloning.
