AllStars Negative Control siRNA

• Most thoroughly validated negative control available
• No homology to any known mammalian gene
• Minimal nonspecific effects
• Can be used in miRNA mimic experiments

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

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.

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.

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

• All routine RNAi experiments
• Start-up RNAi experiments
• High-throughput RNAi screening
• Experiments involving miRNA mimic transfection