Controls for RNAi Experiments

Transfection Controls
Having the highest transfection efficiency possible is the key to good results, particularly for gene knockdown experiments. You will first want to optimize transfection conditions for the cell lines you are working with, so you know you’ll have the optimal transfection conditions for effective RNAi experimentation. Keep in mind that it is also important to monitor experiment-to-experiment transfection variation.

With overexpression experiments, even those with low transfection efficiencies, you can often see results. In contrast, for gene knockdown to be measurable in a cell population, it is important to have the highest transfection efficiency as possible. Even small reductions in transfection efficiency can determine whether you can identify functional differences in your experimental samples or if you are able to validate knockdown by qRT-PCR or Western Analysis.

Invitrogen recommends three transfection reagents for delivering standard siRNA or Stealth RNAi™ siRNA to cells: Lipofectamine™ RNAiMAX, Lipofectamine™ 2000, and Oligofectamine™. To monitor transfection efficiency using these reagents, Invitrogen offers two fluorescently labeled RNAi duplexes: the BLOCK-iT™ Alexa Fluor® Red Fluorescent Control for use with Lipofectamine™ RNAiMAX, and the green BLOCK-iT™ Fluorescent Oligo for use with Lipofectamine™ 2000 or Oligofectamine™ (the BLOCK-iT™ Alexa Fluor® Red Fluorescent Control can also be used with Lipofectamine™ 2000 or Oligofectamine™). Using the green control, you can also measure cytotoxity with the Dead Cell Stain in the RNAi Basic Control Kit.

Click here for more information on controls to use for optimizing transfection with synthetic RNAi or a table listing the contents for each of the transfection control kits.

Invitrogen also recommends two transfection reagents for delivering RNAi vectors to cells: Lipofectamine™ 2000 and Lipofectamine™ LTX. We also recommend transfecting a vector that expresses a fluorescent protein to monitor cellular uptake of the RNAi vector (BLOCK-iT™ Pol II miR RNAi Expression Vector with EmGFP).

Click for more information on controls to use for optimizing transfection with RNAi vectors or a table listing the contents for each of the transfection control kits.

Negative controls
Negative controls allow the measurement of the effect of experimental RNAi molecules versus background. Including one or more negative controls in every RNAi experiment ensures that your results are due to your targeted RNAi molecules and not an artifact of the delivery method. By using the BLOCK-iT™ RNAi Designer, you can choose scrambled controls for any siRNA or Stealth RNAi™ siRNA sequence. We also provide a negative control in our RNAi vector cloning kits. The negative control should be of the same chemical structure as the target RNAi molecules. For example, if you are using shRNA vectors, the negative control should have the same vector backbone, but a different RNAi sequence. For experiments using Stealth RNAi™ siRNA, we have three predesigned negative controls with the following features:

• Three levels of GC content to match that of the experimental Stealth RNAi™ siRNA
• No homology to any known vertebrate gene
• Tested sequences, which do not induce stress response

We recommend using one or more negative control in every RNAi experiment.

Positive controls
Positive controls provide more confidence in your RNAi experiments by ensuring that the experimental conditions were met to achieve robust data. Positive controls are RNAi molecules that are known to achieve high levels of knockdown (>70%). A positive control should be used to optimize RNAi delivery conditions and then reconfirm high levels of delivery in each RNAi experiment. When a positive control fails to produce the anticipated phenotype, carefully evaluate your experimental conditions and decide if some factors need to be adjusted. Positive controls can be genes expressed at easily detectable levels, such as p53, lamin or GAPDH. If looking at a particular phenotype such as apoptosis, you will most likely want to target a gene known to elicit apoptosis, such as EG5.

Downstream controls
Before transfecting cells and performing qRT-PCR and Western Blots to measure mRNA and protein levels, respectively, we recommend validating with downstream reagents. Validating qRT-PCR primers or antibodies for your positive control and target genes before performing knockdown experiments in your cell line ensures that the genes are expressed at a high enough level to interpret the knockdown results. If the genes are not expressed at high enough levels (RNAi Basic Control Kit).

Interferon controls
The introduction of RNAi reagents to cells can induce cellular stress response pathways, such as the interferon response. Activation of these stress response pathways can lead to translational arrest, growth inhibition, and cellular toxicity. These events make it difficult to assess whether observed cellular phenotypes are due to non-specific stress responses or the loss of function of the targeted gene. Validated qRT-PCR primers for PKR, IFIT-1 and 5’OAS stress response genes provide a specific and sensitive way to monitor whether toxic cellular effects are complicating the interpretation of your RNAi experimental data.

Multiple RNAi sequences to the same target gene
RNAi sequences with partial homology to other targets may contribute to off-target activity. Gene profiling experiments have shown that duplexes with partial homology to other transcripts can cleave the target or act like a micro RNA (miRNA), inhibiting translation of the target mRNA. Specificity studies have revealed that siRNA duplexes can have varying activities depending on the number, position, and base pair composition of mismatches with respect to the target RNA. To ensure that knockdown of the intended gene causes a particular RNAi phenotype, the phenotypic results should be confirmed by at least two RNAi molecules that target non-overlapping regions of the target mRNA. Thus, if one RNAi sequence produces a particular phenotype, but the second RNAi sequence (designed to target the same gene) produces a different phenotype, then you cannot conclude that the gene of interest was successfully knocked down.

Titration of RNAi
Stealth RNAi™ siRNA and siRNA can be very effective even at low concentrations. By titrating down the dose of the Stealth RNAi™ siRNA or siRNA duplex you can reduce any off-target or non-specific effects while achieving robust knockdown.

Rescue experiments
RNAi rescue experiments are performed to ensure that the observed effect is due to knocking down the target gene of interest. If using an inducible RNAi vector system, turn off the RNAi expression by removing tetracycline from the medium. If using siRNA or Stealth RNAi™ siRNA there are two main methods used to rescue the phenotype. The first method involves designing RNAi sequences to the 3’UTR and then transfecting the cells after knockdown with a vector expressing the gene of interest, the open reading frame (ORF). If the RNAi sequences were designed to the ORF, you can use a mutagenesis kit to create a one or more silent third-codon point mutations within the region targeted by the RNAi sequence, preferably the seed and cleavage regions on the antisense strand (bases 2-12).