RNA interference (RNAi) has become a powerful technique for studying gene function, biological pathways, and the physiology of diseases. Typically, the RNAi response in mammalian cells is mediated by small interfering RNA (siRNA). The use of synthesized siRNA to silence gene is relatively quick and easy, but it is costly with transient effects. A short hairpin RNA (shRNA) with complementary sense and antisense sequences of a target gene separated by a loop structure results in gene silencing that is as effective as chemically synthesized siRNA with fewer limitations. However, current methods for constructing shRNA vectors require the synthesis of long oligonucleotides, which is costly and often suffers from mutation problems during synthesis. Here, we report an alternative approach to generate a shRNA expression vector with high efficacy. We utilized shorter (<or=50-nucleotide) primers to generate a shRNA insert by the primer extension method. Our new approach for the construction of shRNA expression vectors dramatically reduced the possibility of mutations. Using this method, we constructed a microRNA (miRNA) library, which facilitates the expression of 254 matured miRNAs. We also performed high-throughput screening of miRNAs involved in the regulation of human Survivin promoter activity in lung A549 cells. We found that the expression of miR-192, 199a, 19a, 20a, 213, and 371 caused the activation of the Survivin promoter whereas miR-302b*, 34a, 98, 381, 463 and 471 decreased the Survivin promoter activity.