Criteria for effective design, construction, and gene knockdown by shRNA vectors

The use of mixed oligonucleotide pairs provides a time and cost efficient method of producing wild type and mutant control shRNA vectors. The addition to sequencing reactions of a combination of mixed dITP/dGTP chemistries and DNA relaxing agents enables read through the intrinsic secondary structure of problematic shRNA vectors. Six published algorithms for siRNA oligonucleotide design that were tested in this study show little or no efficacy at predicting shRNA knockdown outcome. However, application of a modification based on the central shRNA stability should provide a useful improvement to the design of effective shRNA vectors.