Using the right controls in each and every RNAi experiment can speed up your research, streamline analysis, and give you confidence in your results. Invitrogen 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
stain
Nuclei
stain
Positive, negative, & reporter controls We recommend
BLOCK-iT™ Fluorescent Oligo
(2013
13750062)
Green BLOCK-iT™ Fluorescent Oligo    
BLOCK-iT™ Alexa Fluor® Red Fluorescent Control
(14750100)
BLOCK-iT™ Alexa Fluor® Red Fluorescent Control    
Dead Cell Stain
(E1169)
 ethidium homodimer-1 (EthD-1)   

Nuclei Stain
(H3570)

  Hoechst 33342  
BLOCK-iT™ Transfection Kit
(13750070)
Green BLOCK-iT™ Fluorescent Oligo   Lipofectamine™ 2000 Transfection Reagent
BLOCK-iT™ Transfection Optimization Kit (human)
(13750047)
Green BLOCK-iT™ Fluorescent Oligoethidium homodimer-1 (EthD-1)Hoechst 33342Scrambled Stealth RNAi™ siRNA duplex (negative)
Human p53 Stealth RNAi™ siRNA duplex (positive)
 
BLOCK-iT™ RNAi Basic Control kit (human)
(K4911-00)
Green BLOCK-iT™ Fluorescent Oligoethidium 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
(12935-200)
   Scrambled Stealth RNAi™ siRNA duplex (36% GC) (negative) 
Stealth RNAi™ siRNA negative control med GC
(12935-300)
   Scrambled Stealth RNAi™ siRNA duplex (48% GC) (negative) 
Stealth RNAi™ siRNA negative control hi GC
(12935-400)
   Scrambled Stealth RNAi™ siRNA duplex (68% GC) (negative) 
Stealth RNAi™ siRNA negative control kit
(12935-100)
   Kit containing the lo, med and hi GC Scrambled Stealth RNAi™ siRNA control duplexes (negative) 
Stealth RNAi™ siRNA GAPDH Positive Control (human)
(12935-140)
    Stealth™ GAPDH duplex (positive) 
Stealth RNAi™ siRNA Actin Positive Control (human) 
(12935-141)
    Stealth™ Actin duplex (positive) 
Stealth RNAi™ siRNA Cyclophilin B Positive Control (human)
(12935-142)
    Stealth™ Cyclophilin duplex (positive) 
Stealth RNAi™ siRNA GFP Reporter Control
(12935-145)
    RNAi GFP Reporter duplex (reporter) 
Stealth RNAi™ siRNA Luciferase Reporter Control
(12935-146)
    RNAi Luciferase Reporter duplex (reporter) 
Stealth RNAi™ siRNA B-lactamase Reporter Control
(12935-148)
    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 OligoGreen BLOCK-iT™ Fluorescent Oligo     2013
13750062
BLOCK-iT™ Alexa Fluor® red fluorescent controlBLOCK-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 kitGreen BLOCK-iT™ Fluorescent Oligo    Lipofectamine™ 200013750070
Scrambled negative control   Custom design your scrambled siRNA control using the BLOCK-iT™ RNAi Designer  BLOCK-iT™ RNAi Designer