QIAGEN AllStars RNAi Controls

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.