Generation of three kinds of promoter-shRNA expressioncassettes and their application in screening of efficient siRNA

For the purpose of rapid screening of efficient siRNA, one-step overlapping extension polymerase chain reaction(PCR)strategy with specific extension primer, upstream (universal) primer and downstream (universal) primer, was developed to promptly and economically prepare small hairpin RNA (shRNA) expression cassettes (SECs) containing three kinds of RNA polymerase III (RNA Pol III) promoter, U6+1, H1 or tRNA~(val).Flow cytometric analysis showed that transfection of PCR-based enhanced green fluorescent protein (EGFP)-specific SECs into HepG2 cells resulted in inhibition of transient expression of EGFP, and that tRNA~(Val)-SEC demonstrated much more inhibition effect compared with U6+1-and H1-SEC in HepG2 cells. Furthermore, no interferon-induced 2'5'OAS mRNA expression was detected in any of the SEC-transfected cells. The results indicated that the SECs could be transfected into cells effectively to induce RNA interference (RNAi), and the PCR-based SEC strategy can be applied in identification of optimal siRNA and its matching promoters.