Synthesis of Poly Linear shRNA Expression Cassettes Through Branch-PCR.

A facile and universal strategy to construct the poly linear small hairpin RNA (shRNA) expression cassettes with multiple shRNA transcription templates through polymerase chain reaction with flexible branched primers (branch-PCR) is described in this protocol. Double-stranded RNA (dsRNA) is not stable enough for the study of RNA interference (RNAi) delivery in mammalian cells. Therefore, the more stable shRNA transcription template is employed to produce the endogenous transcribed dsRNA. Then, the covalent crosslinked linear shRNA expression cassettes are constructed through the branch-PCR for the long-lasting RNAi effect in this protocol. The branched primer pair is efficiently synthesized through classic click chemistry. In one step of PCR, the much more stable poly linear shRNA expression cassettes can be produced in large scale. This strategy of efficient synthesis of the poly linear gene expression cassettes can also be applied in the field for other target gene delivery. © 2016 by John Wiley & Sons, Inc.