The effect of silencing ARPC1B on cell migration was studied in a breast cancer cell line. A monolayer of cells was uniformly scraped, and the rate of cell migration to close the scrape (wound healing) was evaluated. Both unmodified and ON-TARGETplus siRNA reagents induced potent target knockdown. Inconsistent phenotypes due to off-target effects (red outline) were observed for cells transfected with unmodified individual siRNAs.
The unmodified SMARTpool improved the false phenotype considerably while the ON-TARGETplus SMARTpool significantly reduced off-target effects to produce a consistent phenotype. In collaboration with Kaylene Simpson, Laura Selfors and Joan Brugge, Harvard Medical School.
Panels (A) and (B) are representative examples of off-target signatures with and without application of ON-TARGETplus modifications to (A) a single siRNA and (B) a SMARTpool reagent. Green bars indicate genes with 2-fold or more reduction of expression when treated with the indicated siRNA reagent.
The ON-TARGETplus modifications reduced the off-targets when compared to unmodified siRNA. Pooling of siRNA and the ON-TARGETplus modification pattern independently and in combination, provide significant reduction in off-target gene silencing.
Panel (C) represents quantitation of off-targets (down-regulated by 2-fold or more) induced by the indicated siRNA reagents targeting 10 different genes (4 siRNAs per gene or a single SMARTpool reagent). Off-targets were quantified using microarray analysis (Agilent), then compiled. Each shaded box represents the middle 50% of the data set. Horizontal line in box: Median value of the data set. Vertical bars: minimum and maximum data values.