Multiple negative control siRNAs (Control 1– Control 10) were transfected in triplicate into MCF-7 cells. After incubation, cRNA was prepared and hybridized to Affymetrix human U133 GeneChip arrays. Regulated genes were identified as genes that showed at least a 1.5-fold change in expression (both upregulated and downregulated) compared to untransfected cells. Ingenuity pathway analysis software was used to determine the proportion of regulated genes in each pathway compared to the total number of genes identified as central to that pathway. Where a bar appears in the figure, this means that genes in the pathway were regulated by the siRNA. If every pathway gene was regulated, the relative proportion would be 100%. Lower bars therefore indicate a lower relative proportion of regulated genes within that pathway. Where no bar appears, no genes of the pathway were regulated by the siRNA. AllStars Negative Control siRNA (indicated with arrow) resulted in the lowest number of regulated genes. In contrast, other control siRNAs resulted in higher numbers of regulated genes from important cellular pathways.|Identical numbers of MCF-7 cells were transfected with AllStars Negative Control siRNA or another negative control siRNA (Control 1). Untransfected cells were also analyzed. After 72 hours, cells were harvested, washed in PBS, transferred to TruCOUNT tubes, and counted using FACS analysis.|
MCF-7 and HeLa cells were cotransfected with the reporter construct and either a noncomplementary siRNA or AllStars Negative Control siRNA. After 24 hours, expression of the fluorescent reporter gene was measured by [A] fluorescence microscopy (HeLa cells shown) and [B], [C] FACS analysis. Normal fluorescence was observed after cotransfection with the noncomplemetary siRNA, showing that the reporter gene is expressed. After cotransfection with AllStars Negative Control siRNA fluorescence was significantly decreased, showing that the reporter gene is downregulated.
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HeLa cells were cotransfected with the reporter construct and either a noncomplementary siRNA or AllStars Negative Control siRNA. After 48 hours, western blot analysis was used for measurement of expression of the His tag. The His tag was expressed after cotransfection with the noncomplementary siRNA. After cotransfection with AllStars Negative Control siRNA, the His tag was not detected.
|HCT-116 cells (6 x 10
4) were
[A] untransfected or transfected with
[B] AllStars Negative Control siRNA or
[C] another negative control siRNA (Control 1). After 72 hours, cells were treated with BrdU, fixed, and permeabilized. Cells were then stained using anti-BrdU–FITC antibody. DNA synthesis rates were measured by determining the percentage of BrdU-positive cells (region M2) using FACS analysis. The percentage of BrdU-positive cells was similar in untransfected cells and cells transfected with AllStars Negative Control siRNA (53.1% and 52.5% respectively). In contrast, cells transfected with Control 1 showed only 10% BrdU-positive cells indicating altered DNA synthesis.|Identical numbers of MCF-7 cells were
[A] untransfected or
[B] transfected with AllStars Negative Control siRNA, or
[C] transfected with another negative control siRNA (Control 7). After 72 hours, all cells from the culture flasks were stained with propidium iodide, counted by FACS, and the number of living (propidium iodide negative) and dead (propidium iodide positive) cells were determined. Transfection of AllStars Negative Control siRNA resulted in similar numbers of dead cells as seen for untransfected cells (10.8% and 10.1% dead cells, respectively). The other control siRNA caused an increase in the level of cell death to 28.7%.|MCF-7 cells were
[A] untransfected or
[B] transfected with AllStars Negative Control siRNA or
[C] CDC2 siRNA. CDC2 siRNA was transfected as a positive control, as knockdown of CDC2 is known to affect the cell cycle and result in accumulation of cells in the G2 phase. After 72 hours, cells were detached from the culture plate using trypsin and fixed in 70% ethanol prior to treatment with RNase and propidium iodide staining. FACS analysis was performed for 40,000 cells of each sample. Cell-cycle distribution for cells transfected with AllStars Negative Control siRNA was similar to that observed for untransfected cells. Cell-cycle distribution for cells transfected with CDC2 siRNA showed accumulation of cells in the G2 phase as expected.|
The reporter construct consisted of artificial target sequence complementary to AllStars Negative Control siRNA fused to a fluorescent reporter gene with a His tag.
|HCT 116 cells were transfected with AllStars Negative Control siRNA, another negative control siRNA (Control 1), and CDC2 siRNA. CDC2 siRNA was transfected as a positive control, as knockdown of CDC2 is known to affect the cell cycle and result in enlarged nuclei. Untransfected cells were also analyzed. After 72 hours, the nuclei of live cells were stained with Hoechst 33342 and the surface area of individual cell nuclei was measured using the cell^R Imaging System (Olympus). In this graph, 100 nuclear size measurements per treatment were plotted in order of size. AllStars Negative Control siRNA resulted in a nuclear size profile similar to untransfected cells. CDC2 siRNA resulted in enlarged nuclei as expected. The negative control siRNA, Control 1, also resulted in enlarged nuclei.|
