QIAGEN AllStars RNAi Controls

QIAGEN AllStars RNAi Controls
Extensively characterized controls for RNAi in human, mouse, and rat
On the QIAGEN AllStars RNAi Controls web page, you can easily find out what control experiments you need to run, and search for and order the appropriate QIAGEN AllStars RNAi Control. Browse the sections below to choose the right controls to ensure that your RNAi data is correctly interpreted and analyzed.
AllStars Positive Controls — siRNAs to control for optimal conditions
AllStars Negative Controls — highly validated nonsilencing siRNA 
AllStars Transfection Controls — siRNAs for assessment of transfection efficiency
Untransfected control — analysis of untreated cells
AllStars Positive Controls — siRNAs to control for optimal conditions
Positive control siRNAs to run in every RNAi experiment:
  • During start-up experiments, a positive control siRNA can be used to determine optimal conditions.
  • A positive control siRNA transfected in every experiment will indicate if conditions become suboptimal.
Commonly used controls
Positive control siRNAs have been proven to cause high knockdown of their target gene. siRNAs targeting ubiquitous and highly expressed housekeeping genes are commonly used controls. After transfection in the control experiment, knockdown can be assessed by, for example, quantitative real-time RT-PCR.
Cell death control
A cell death control causes cell death that can be observed by light microscopy. Highly potent AllStars Hs Cell Death Control siRNA (for human cells) or AllStars Mm/Rn Cell Death Control siRNA (for mouse or rat cells) are siRNA blends that target ubiquitous cell survival genes. These siRNA blends have been functionally validated in a wide range of cell lines and primary cells. Transfection of AllStars Hs Cell Death Control siRNA into human cells or of AllStars Mm/Rn Cell Death Control siRNA into mouse or rat cells results in a high degree of cell death that is visible by light microscopy, making these siRNA blends invaluable tools for siRNA transfection optimization and routine positive control experiments (see Fast and easy analysis of mouse and rat cells and Primary human NHBE cells after transfection of AllStars Hs Cell Death Control siRNA).
Key pathway gene controls
Key pathway gene controls are siRNAs targeting genes central to important pathways, for example, kinases or genes involved in cellular signaling or apoptosis. If you are working with genes of a particular family or are interested in the biology of a specific pathway (e.g., if using an assay detecting apoptosis), you may want to knockdown a key pathway gene as a positive control (see figure AllStars Control induces high level of cell death).
Principle and procedure
Positive control siRNA should be routinely transfected in every experiment to ensure optimal conditions are maintained. The positive control siRNA should always result in high knockdown of the target gene as measured by quantitative or phenotypic analysis. If high knockdown is not achieved, this indicates a problem with the experimental setup. Positive control siRNAs also enable RNAi start-up experiments in which multiple transfections are performed to determine optimal conditions. In addition, in high-throughput RNAi screens, AllStars Cell Death Control siRNAs are ideal for use as internal positive controls to ensure that optimal transfection conditions are maintained on every plate.

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AllStars Negative Controls — highly validated nonsilencing siRNA
Nonsilencing siRNAs to run in every RNAi/miRNA experiment:
  • A negative control will indicate if changes in phenotype or gene expression are nonspecific.
Product description
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. It has been validated using Affymetrix GeneChip arrays and a variety of cell-based assays and shown to ensure minimal nonspecific effects on gene expression and phenotype (see table below). 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 (see figure Low nonspecific effects on expression). 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. In addition, due to the similarity between siRNA and miRNA molecules, AllStars Negative Control siRNA can be used as a negative control in experiments involving transfection of miRNA mimics.

Advantages of using thoroughly tested and validated AllStars Negative Control siRNA:
  • Results from AllStars Negative Control siRNA can be compared to results from untransfected cells to determine whether the experimental setup causes nonspecific effects.
  • Results from AllStars Negative Control siRNA can be compared to results from gene-specific siRNA to pinpoint the effects of target gene knockdown.
  • In miRNA mimic experiments, results from AllStars Negative Control siRNA can be compared to results from gene-specific miRNA mimics to pinpoint the effects of target downregulation (see Guidelines for miRNA mimic and miRNA inhibitor experiments).
Principle and procedure
It is essential to transfect a negative control siRNA in every experiment. Results from the negative control should be compared to 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, i.e., 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 to 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.
Data for AllStars Negative Control siRNA
The following table shows the tests performed on a range of negative control siRNAs of different types including nonsilencing siRNAs, scrambled siRNAs, and siRNAs targeting artificial reporter genes. AllStars Negative Control siRNA consistently provided optimal results. To view data from each test, click on the link in the table.

Test performed
Test name Tested for Best result for AllStars Negative Control siRNA (compared to untreated cells) Data
Genomewide analysis to test for nonspecific effects on expression
(MCF-7, K562, and HUVEC cells)
Affymetrix GeneChip arrays Nonspecific regulation of gene expression Minimal number of genes regulated Low nonspecific effects on expression
Live-cell nuclei staining Nuclear size Normal No affect on nuclear size phenotype
Cell number Proliferation rates Unchanged AllStars Negative Control siRNA does not affect cell number
Nucleotide incorporation DNA synthesis rates Unchanged Normal DNA synthesis phenotype
Live-cell dye exclusion Cytotoxic effects Unchanged No cytotoxic effects
DNA staining Cell-cycle distribution Normal Normal cell-cycle distribution
RISC-incorporation analysis (HeLa and MCF-7 cells)
Reporter construct transfection Determine if siRNA is incorporated into RISC (a valid negative control siRNA should enter RISC) Incorporated into RISC AllStars Negative Control siRNA is incorporated into RISC and Western blot analysis shows AllStars Negative Control siRNA enters RISC

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AllStars Transfection Controls
Fluorescently labeled controls
AllStars Transfection Controls include siRNAs which are fluorescently labeled on the 3' end of the sense strand such as AllStars Negative Control siRNA, which is available with a variety of fluorescent label options, or AllStars Cell Death Control siRNA (see Fast and easy analysis of mouse and rat cells and Primary human NHBE cells after transfection of AllStars Hs Cell Death Control siRNA). 

If a fluorescent label on a functional siRNA is required, FlexiTube siRNA and Custom siRNA Synthesis are available with modification options.
Principle and procedure
When establishing RNAi in start-up experiments or in a new cell line, it is necessary to perform multiple transfections under different conditions to determine the optimal conditions for maximum transfection efficiency (see figures Primary human NHBE cells after transfection of AllStars Hs Cell Death Control siRNA and Easy siRNA transfection optimization of MCF-7 cells). These optimization experiments can be performed using AllStars Transfection Controls. Transfection conditions that result in the greatest percentage of fluorescent cells (if using a fluorescently labeled control) or cell death (if using a cell death control) should be maintained in future experiments.

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Untransfected control
Untransfected cells should be routinely analyzed in every experiment
  • Analysis of an untransfected control shows the gene expression levels in the absence of any treatment.
  • Data from the untransfected control can be compared to data from transfection of gene-specific siRNA/miRNA to calculate relative target gene expression.
Principle and procedure
Downstream analysis to determine gene expression (e.g., real-time RT-PCR or western blot analysis) is performed on samples after transfection of siRNA or miRNA mimic/inhibitor and also on untransfected samples which have received no treatment.

Normalized gene expression in the untransfected control should be compared to that from transfection of the negative control siRNA/miRNA. Gene expression should be similar in both untransfected cells and cells transfected with negative control. Any differences in gene expression between these two samples are caused by nonspecific effects (for example, see figure Allstars Control induces high level of cell death). 

Gene expression in samples transfected with functional gene-specific siRNA/miRNA may be calculated relative to the untransfected control.


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Fast and easy analysis of mouse and rat cells
Fast and easy analysis of mouse and rat cells.
Transfected with various amounts of AllStars Mm/Rn Cell Death Control siRNA or 25 nM nonsilencing siRNA (AllStars Negative Control siRNA) using HiPerFect Transfection Reagent. After 72 hours, cell death was observed by light microscopy. All conditions used induced a cell-death phenotype that could easily be distinguished from the nonsilencing control.
Primary human NHBE cells after transfection of AllStars Hs Cell Death Control siRNA
Primary human NHBE cells after transfection of AllStars Hs Cell Death Control siRNA.
Cells (2 x 104) were transfected with 10 nM, 20 nM, or 100 nM AllStars Hs Cell Death Control siRNA or 100 nM nonsilencing siRNA (AllStars Negative Control siRNA) using HiPerFect Transfection Reagent. After 48 hours, cell death was observed by light microscopy.
AllStars Control Induces High Level of Cell Death
AllStars Control induces high level of cell death
[A] SW620 cells or [B] SW480 cells (5 x 103 cells per well in 96-well plates) were transfected with 20 nM of AllStars Hs Cell Death Control siRNA or AllStars Negative Control siRNA or an siRNA targeting PLK (a well-known target for induction of cell death). Untransfected cells and cells treated with transfection reagent only were also analyzed. After 72 hours, cell death was quantified using a CellTiter-Blue assay (Promega). AllStars Hs Cell Death Control siRNA resulted in high levels of cell death in both cell types.

(Data kindly provided by Dr. Amanda Hummon and Dr. Thomas Reid, National Cancer Institute, Maryland, U.S.A.)
Low Nonspecific Effects on Expression Using AllStars Negative Control siRNA
Low Nonspecific effects on expression.
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.
AllStars Negative Control siRNA Does Not Affect Nuclear Size Phenotype
No affect on nuclear size phenotype.
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.
AllStars Negative Control siRNA Does Not Affect Cell Number
AllStars Negative Control siRNA does not affect cell number.
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.
Normal DNA Synthesis Phenotype Using AllStars Negative Control siRNA
Normal DNA synthesis phenotype.
HCT-116 cells (6 x 104) were transfected with AllStars Negative Control siRNA or another negative control siRNA (Control 1). Untransfected cells were also analyzed. 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.
AllStars Negative Control siRNA does not cause cytotoxic effects
No cytotoxic effects.
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%.
Normal Cell-Cycle Distribution
Normal cell-cycle distribution.
MCF-7 cells were untransfected or transfected with AllStars Negative Control siRNA or 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.
AllStars Negative Control siRNA is Incorporated into RISC
AllStars Negative Control siRNA is incorporated into RISC.
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]ACS 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.
Western analysis shows AllStars Negative Control siRNA enters RISC
Western analysis shows AllStars Negative Control siRNA enters RISC.
HeLa cells were cotransfected with the reporter construct and either a noncomplementary siRNA or AllStars Negative Control siRNA. After 48 hours, Wstern 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.
Easy siRNA Transfection Optimization MCF-7 cells
Easy siRNA transfection optimization of MCF-7 cells.
Easy siRNA Transfection Optimization MCF-7 cells (5 x 103) in 96-well plates were transfected with various amounts of AllStars Hs Cell Death Control siRNA or 50 nM nonsilencing siRNA (AllStars Negative Control siRNA) using HiPerFect Transfection Reagent. After 72 hours, cell death was observed by light microscopy. Optimal conditions were 5 nM siRNA and 1.5/2 μl HiPerFect Reagent or 2 nM siRNA and 2 μl HiPerFect Reagent.