Using the right controls in each and every RNAi experiment can speed up your research, streamline analysis, and give you confidence in your results. Life Technologies has a wide selection of controls for your RNAi experiments.
Highly efficient delivery of Stealth RNAi™ siRNA or standard siRNA is necessary for significant levels of knockdown. Optimizing transfection, controlling for experimental variability, and using a powerful transfection reagent all raise the likelihood of RNAi success. The use of positive and negative controls may also help in assessing your experiments.
Optimizing transfection conditions
Good results in any RNAi experiment depend upon high transfection efficiency. Before starting RNAi experiments in a new cell line, we recommend optimizing transfection conditions. Invitrogen has a number of free transfection protocols on our website, but each assay is different and you will probably need to adjust the protocol to your needs.
For measurable gene knockdown in a cell population, it is very important to have the highest transfection efficiency. Even small reductions in transfection efficiency can determine whether you identify functional differences in your experimental samples.
Controls for optimizing transfection conditions:
- Fluorescently tagged oligonucleotide to determine that the RNAi duplex has entered the cell
- Dead cell stain to monitor transfection toxicity
- Nuclei stain to measure the percent of transfected and/or dead cells
- Positive and negative Stealth RNAi™ siRNA or standard siRNA controls to measure the knockdown levels in each transfection condition.
See below for a table of Invitrogen kits containing these 4 controls.
Once you have optimized the transfection conditions, we also recommend using these four types of controls in every RNAi experiment to monitor experiment-to-experiment transfection variation. For more information on how on to use controls and plan RNAi experiments, please read Invitrogen’s Seven Steps Toward RNAi Success.
Table 1 - Controls for Stealth RNAi™ siRNA experiments
Table 1 lists all of the available Invitrogen Kits that contain controls for transfections using Stealth RNAi™ siRNA. Controls 1-4 refer to the controls outlined in the numbered bullets above and are recommended for use in every RNAi experiment.
|Product Name||Kit Contents|
|Fluorescently tagged oligo||Dead cell
|Positive, negative, & reporter controls||We recommend|
|BLOCK-iT™ Fluorescent Oligo
|Green BLOCK-iT™ Fluorescent Oligo|
|BLOCK-iT™ Alexa Fluor® Red Fluorescent Control
|BLOCK-iT™ Alexa Fluor® Red Fluorescent Control|
|Dead Cell Stain
|ethidium homodimer-1 (EthD-1)|
|BLOCK-iT™ Transfection Kit
|Green BLOCK-iT™ Fluorescent Oligo||Lipofectamine™ 2000 Transfection Reagent|
|BLOCK-iT™ Transfection Optimization Kit (human)
|Green BLOCK-iT™ Fluorescent Oligo||ethidium homodimer-1 (EthD-1)||Hoechst 33342||Scrambled Stealth RNAi™ siRNA duplex (negative)
Human p53 Stealth RNAi™ siRNA duplex (positive)
|BLOCK-iT™ RNAi Basic Control kit (human)
|Green BLOCK-iT™ Fluorescent Oligo||ethidium homodimer-1 (EthD-1)||Scrambled Stealth RNAi™ siRNA duplex (negative)
Human p53 Stealth RNAi™ siRNA duplex (positive)
|Lipofectamine™ 2000 Transfection Reagent,
LUX™ Primer sets: human p53 and human GAPDH
|Stealth RNAi™ siRNA negative control lo GC
|Scrambled Stealth RNAi™ siRNA duplex (36% GC) (negative)|
|Stealth RNAi™ siRNA negative control med GC
|Scrambled Stealth RNAi™ siRNA duplex (48% GC) (negative)|
|Stealth RNAi™ siRNA negative control hi GC
|Scrambled Stealth RNAi™ siRNA duplex (68% GC) (negative)|
|Stealth RNAi™ siRNA negative control kit
|Kit containing the lo, med and hi GC Scrambled Stealth RNAi™ siRNA control duplexes (negative)|
|Stealth RNAi™ siRNA GAPDH Positive Control (human)
|Stealth™ GAPDH duplex (positive)|
|Stealth RNAi™ siRNA Actin Positive Control (human)
|Stealth™ Actin duplex (positive)|
|Stealth RNAi™ siRNA Cyclophilin B Positive Control (human)
|Stealth™ Cyclophilin duplex (positive)|
|Stealth RNAi™ siRNA GFP Reporter Control
|RNAi GFP Reporter duplex (reporter)|
|Stealth RNAi™ siRNA Luciferase Reporter Control
|RNAi Luciferase Reporter duplex (reporter)|
|Stealth RNAi™ siRNA B-lactamase Reporter Control
|RNAi B-lactamase duplex (reporter)|
|Scrambled negative control
(BLOCK-iT™ RNAi Designer )
|Custom scrambled Stealth RNAi™ siRNA control (negative) using BLOCK-iT™ RNAi Designer|
Table 2 - Controls for standard siRNA Experiments
Table 2 contains all of the available Invitrogen Kits that contain controls for transfections using standard siRNA. Controls 1-4 refer to the controls outlined in the numbered bullets above and are recommended for use in every RNAi experiment.
|Product Name||Kit Contents||Cat. No.|
|Control 1) Flur. Tagged Oligo||Control 2) Dead Cell Stain||Control 3) Nuclei Stain||Control 4a) Negative Control||Control 4b) Positive Control||Transfection Reagent|
|BLOCK-iT™ Fluorescent Oligo||Green BLOCK-iT™ Fluorescent Oligo||2013
|BLOCK-iT™ Alexa Fluor® red fluorescent control||BLOCK-iT™ Alexa Fluor® red fluorescent control||14750-100|
|Dead cell stain||ethidium homodimer-1 (EthD-1)||E1169|
|Nuclei Stain||Hoechst 33342||H3570|
|BLOCK-iT™ Transfection kit||Green BLOCK-iT™ Fluorescent Oligo||Lipofectamine™ 2000||13750070|
|Scrambled negative control||Custom design your scrambled siRNA control using the BLOCK-iT™ RNAi Designer||BLOCK-iT™ RNAi Designer|