shRNA structure-activity analysis. (A) Distribution of shRNA activities according to the Dharmacon score and shRNA orientation. Closed circles, SA shRNAs. Open circles, AS shRNAs. (B) Activity of shRNAs that passed (closed circles) or failed (open circles) the combination of five filtering criteria but not target disruption energy (Top) or all six filters combined (Bottom). P values were determined by Welch's t test (two sided, unequal variance). Left: luciferase-derived shRNA. Right: cDNA-derived shRNA.

Selection and validation of growth-inhibitory shRNAs. (A) Scheme of BrdU suicide selection for doxycycline-dependent growth inhibition. (B) Scheme of selection and analysis of growth-inhibitory shRNAs. (C) Testing 22 gene targets enriched by shRNA selection. Four synthetic siRNAs per target (sets A–D) from Qiagen were tested for the ability to inhibit MDA-MB-231 cell growth. A cytotoxic siRNA mixture (tox) was used as a positive control; siRNAs targeting GFP (siGFP) or targeting no human gene (siControl) were used as negative controls. (D) The same analysis conducted on 12 gene targets with unaltered shRNA representation after BrdU suicide selection.

shRNA library construction. (A) Scheme of shRNA library construction from randomly fragmented DNA. (B) Diagram of LLCEP TU6LX vector. CMV + LTR, LTR promoter with CMV enhancer; SIN LTR, self-inactivating LTR promoter; CPPT, central polypurine tract sequence; WPRE, woodchuck hepatitis posttranscriptional regulatory element; TetOx7, module of 7 tet operator repeats; CAT, chloramphenycol acetyltransferase; ccdB, cytotoxic protein (negative selection marker). (C) RNA expression levels in MCF7 cells, plotted in the order of increasing expression for all human UniGene entries and for genes identified in the shRNA library.

shRNA activity testing. (A) RNAi activity of 230 luciferase-derived shRNAs in luciferase-expressing HT1080 cells, transduced and analyzed in 96-well plates. Target knockdown is measured by reduction in normalized luciferase activity in doxycycline-treated relative to untreated cells. shRNA sequences arranged in the order of increasing activity. Mean and standard deviation shown for triplicate experiments. (B) RNAi activity of 96 luciferase-derived shRNAs from the set in A, retransduced in 24-well plates. Target knockdown is measured by reduction in normalized luciferase activity in doxycycline-treated cells relative to control vector-transduced cells. Knockdown by reference luciferase shRNA (from Clontech) is marked with a dashed line. Mean and standard deviation are shown for duplicate experiments. (C) RNAi activity of 101 cDNA-derived shRNAs in MCF7 cells, transduced and analyzed in 96-well plates. Target knockdown is measured by reduction in RNA levels for the corresponding genes, as measured by QPCR, in doxycycline-treated relative to untreated cells. Mean and standard deviation shown for triplicate QPCR assays. (D) Immunoblotting analysis of the indicated proteins in MCF7 cells transduced with shRNAs targeting the corresponding genes and grown in the absence (-) or in the presence of doxycycline (+). Beta-actin was used as a loading control. RNA knockdown was determined by QPCR; protein knockdown was determined by image quantitation.