AllStars Negative Control siRNA

For negative control RNAi experiments

  • Most thoroughly validated negative control available
  • No homology to any known mammalian gene
  • Minimal nonspecific effects
  • Can be used in miRNA mimic experiments
AllStars Negative Control siRNA is the most thoroughly tested and validated negative control siRNA currently available. This siRNA has no homology to any known mammalian gene. Validation has been performed using Affymetrix GeneChip arrays and a variety of cell-based assays to ensure minimal nonspecific effects on gene expression and phenotype. Minimal nonspecific effects ensure that comparison of the gene-specific siRNA to the negative control gives a true picture of the effects of target-gene knockdown on gene expression and phenotype. If the negative control causes nonspecific effects then results from RNAi experiments can be misleading and difficult to interpret. Cloning experiments confirmed that AllStars Negative Control siRNA enters RISC. AllStars Negative Control siRNA is patent-pending and the sequence is proprietary.
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Thoroughly tested and validated nonsilencing siRNA with no homology to any known mammalian gene,
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The AllStars Negative Control siRNA is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Low nonspecific effects on expression.|Cell number unaffected.|AllStars Negative Control siRNA is incorporated into RISC.|Western analysis shows AllStars Negative Control siRNA enters RISC.|Normal DNA synthesis phenotype.|No increase in cytotoxic effects.|Normal cell-cycle distribution.|Reporter construct for RISC-incorporation experiment|Nuclear size phenotype unaffected.|
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 104) 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.|
Performance

The performance of AllStars Negative Control siRNA was validated by experiments shown in the table.

Tests performed
Test type Test name Purpose Result for AllStars Negative Control siRNA
Genomewide analysis Affymetrix GeneChip arrays Nonspecific regulation of gene expression Minimal number of genes regulated
Cell-based assay Live-cell nuclei staining Nuclear size Normal
Cell-based assay Cell number Proliferation rates Unchanged
Cell-based assay Nucleotide incorporation DNA synthesis rates Unchanged
Cell-based assay Live-cell dye exclusion Cytotoxic effects Unchanged
Cell-based assay DNA staining Cell-cycle distribution Normal
RISC-incorporation analysis (HeLa and MCF-7 cells) Reporter construct transfection Determine whether siRNA is incorporated into RISC (a valid negative control should enter RISC) Incorporated into RISC
Affymetrix GeneChip arrays

Genomewide analysis was performed to test the level of nonspecific effects on gene expression after transfection of multiple negative control siRNAs. Multiple negative control siRNAs from different origins were transfected into MCF-7, K562, and primary HUVEC cells using HiPerFect Transfection Reagent. These included nonsilencing siRNAs (with no homology to mammalian genes), scrambled siRNAs (siRNAs with the same base composition as the gene-specific siRNA but with an altered sequence), and siRNAs targeting artificial reporter genes. Subsequently, expression profiling of the whole human genome was performed with Affymetrix GeneChip arrays. AllStars Negative Control siRNA consistently resulted in the lowest number of nonspecifically regulated genes, making it a highly suitable negative control. In contrast, other negative control siRNAs resulted in nonspecific regulation of many genes from important cellular pathways (see figure "Low nonspecific effects on expression").

Live-cell nuclei staining

Live-cell nuclei staining was used to measure nuclear size. Changes in size could be an indication of cell-cycle disturbance or growth inhibition. A range of negative control siRNAs of different types were tested. AllStars Negative Control siRNA provided the best results of all the controls tested. Transfection of AllStars Negative Control siRNA did not result in any change in nuclear size compared with untransfected cells. In contrast, transfection of another negative control siRNA (Control 1) resulted in enlarged nuclei (see figure "Nuclear size phenotype unaffected").

Cell number

Cell number was assessed after transfection of a range of negative control siRNAs to determine whether cells were proliferating normally. Almost no difference in cell number was observed between untransfected cells and cells transfected with AllStars Negative Control siRNA. In contrast, cell number was significantly reduced after transfection with other negative control siRNAs tested, such as Control 1, indicating that these siRNAs resulted in a growth defect phenotype (see figure "Cell number unaffected").

Nucleotide incorporation

Nucleotide incorporation was measured to determine DNA synthesis rates in untransfected HCT-116 cells and in HCT-116 cells transfected with a range of different negative control siRNAs. Nucleotide incorporation was measured by examining the uptake of Bromodeoxyuridine (BrdU), a base analog of thymidine that substitutes for thymidine during DNA replication and is incorporated into newly synthesized DNA. Changes in DNA synthesis rates could indicate altered cell growth or cell cycle. Cells transfected with AllStars Negative Control siRNA showed a BrdU-incorporation rate that was very similar to that of untransfected cells. However, another negative control siRNA tested (Control 1) resulted in an altered profile with a lower level of DNA synthesis, which indicates that this siRNA affects cell growth or the cell cycle (see figure "Normal DNA synthesis phenotype").

Live-cell dye exclusion

Live-cell dye exclusion was used to measure potential cytotoxic effects of a range of negative control siRNAs. Results showed that cells transfected with AllStars Negative Control siRNA and untransfected cells had a similar number of living and dead cells. In contrast, other negative control siRNAs resulted in an increase in cytotoxicity (see figure "No increase in cytotoxic effects").

DNA staining for cell-cycle analysis

DNA staining after cell fixation was used to measure cell-cycle distribution (the amount of cells in G1/G0, S, and G2 phases of the cell cycle). After transfection of AllStars Negative Control siRNA, the proportion of cells in each phase was similar to that observed for untransfected cells (see figure "Normal cell-cycle distribution"). This result demonstrates that AllStars Negative Control siRNA does not adversely affect the cell cycle.

Reporter construct transfection to assess incorporation into RISC

For accurate negative control RNAi experiments, the negative control siRNA should be incorporated into RISC (RNA-Induced Silencing Complex). This means that the control siRNA goes through the same biological process as the gene-specific siRNA and enables comparison of data from gene-specific siRNA with data from negative control siRNA to confidently determine results that are due to target gene knockdown.

Experiments were performed as follows.

A reporter construct was generated containing an artificial siRNA-target sequence complementary to the AllStars Negative Control siRNA sequence fused to a fluorescent reporter gene with a His tag (see figure "Reporter construct for RISC-incorporation experiment").
The reporter construct was cotransfected with either AllStars Negative Control siRNA or with a noncomplementary siRNA. Untransfected cells were also analyzed.
Fluorescence microscopy and FACS were performed to detect expression of the fluorescent reporter gene. Western blot analysis was performed to detect expression of the fusion protein via its His tag.

Cotransfection of the reporter construct with a noncomplementary siRNA resulted in strong expression of the fluorescent protein and the His tag. When the construct was cotransfected with AllStars Negative Control siRNA, the siRNA knocked down expression from its complementary sequence, resulting in degradation of the entire mRNA transcript encoding the fluorescent reporter gene, the His tag, and the siRNA target sequence. The mRNA degradation resulted in knockdown of the fusion protein (see figures "AllStars Negative Control siRNA is incorporated into RISC" and "Western analysis shows AllStars Negative Control siRNA enters RISC"). To achieve the knockdown observed, AllStars Negative Control siRNA must have entered RISC.

Principle

Transfection of a negative control siRNA is essential in every RNAi experiment. Results from the negative control should be compared with results from untransfected cells. Gene expression and phenotype should ideally be similar in both untransfected cells and cells transfected with negative control siRNA. If altered expression or phenotype are observed in cells transfected with negative control siRNA, these changes are nonspecific – they are due to transfection procedures or siRNA toxicity and not sequence complementarity. Nonspecific effects should be minimal to ensure reliable RNAi/miRNA results.

Results from the negative control can also be compared with results from the gene-specific siRNA/miRNA under study. This comparison allows the researcher to pinpoint the effects of target-gene knockdown on gene expression and phenotype, because the negative control sample has undergone the same biological process, with the only difference being the siRNA/miRNA sequence.

Procedure

Results from AllStars Negative Control siRNA can be used as follows:

Compare with results from untransfected cells to determine whether the experimental setup causes nonspecific effects
Compare with results from gene-specific siRNA to pinpoint the effects of target gene knockdown
In miRNA mimic experiments, compare with results from gene-specific miRNA mimics to pinpoint the effects of target downregulation
Applications
All routine RNAi experiments
Start-up RNAi experiments
High-throughput RNAi screening
Experiments involving miRNA mimic transfection
Feature
Specifications
Design Predesigned/validated by Affymetrix GeneChip Array and cell-based assays
Format Tube
Modification Yes
Scale or yield 5 nmol, 20 nmol
Species Human, mouse, rat
Target sequence provided No

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