Polycistronic expression of interfering RNAs from RNA polymerase III promoters

In many RNA silencing applications, there is a benefit to expressing multiple interfering RNAs simultaneously. This can be achieved by using a single RNA polymerase II promoter to express multiple micro(mi)RNA-formatted interfering RNAs that are arranged in a polycistronic cluster, mimicking the organization of naturally clustered, endogenous miRNAs. While RNA pol III promoters are often used to express individual short hairpin (sh) RNAs, we have recently shown that pol III promoters can also be used to drive polycistronic expression of miRNA-formatted interfering RNAs. Here, we present methods for the assembly of polycistronic miRNA expression vectors that use pol III promoters. In addition, we present methods for testing the potency and the level of expression of each of the individual miRNAs encoded in the construct.