Combined activities measured with an all-in-one reporter. (A) All 10 single shRNA plasmids, all 26 combination plasmids, and the extra non-matched shRNA plasmids (singles plus c ontrol c ombinations (c.c.) - see Additional file 1 for control data) were separately transfected with the three all-in-one reporters; the aio sense (to measure specific activity of the intended guide strand), the aio anti-sense (to measure unintended activities from the expected passenger strand), and the non-matched control to measure potential non-specific effects from our combinations. Off-scale values (> 100%, i.e. no activity) are indicated by open circles and text labels where appropriate. (B) All single shRNA and combination plasmids were re-tested with the aio sense reporter using a titrated amount of shRNA or combination plasmid from 400 ng to 1 ng. In this example, the filler plasmid used to maintain a constant amount of DNA per transfection was our base lentivirus (backbone) plasmid, without any cassettes (i.e. competing H1 promoters). Values shown are representative of 2 or more independently repeated experiments.