Lipid Transfection
| Invitrogen has the most complete collection of transfection reagents with exceptional performance for the delivery of siRNA, Stealth™ RNAi, RNAi vectors, DNA oligonucleotides, and RNA, in traditional or difficult-to-transfect cell lines. The mechanism of cationic lipid-mediated transfection originates with the basic structure of cationic lipids, a positively charged head group and one or two hydrocarbon chains. The positive surface charge of the liposomes mediates the interaction of the nucleic acid and the cell membrane, allowing for fusion of the liposome/nucleic acid (“transfection complex”) with the negatively charged cell membrane. The transfection complex is thought to enter the cell through endocytosis. Once inside the cell, the complex must escape the endosomal pathway and diffuse through the cytoplasm. The first step for successful transfections is to choose the best transfection reagent (Table 1) for your application. Invitrogen provides cell specific RNAi transfection protocols for many popular cell lines, but further optimization may be necessary for your particular RNAi experiments. |
| Transfection product | When to use | Key advantages |
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| Lipofectamine™ RNAiMAX | Transfecting
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| Lipofectamine™ 2000 | Transfecting
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| Oligofectamine™ Transfection Reagent | Transfecting
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| Lipofectamine™ LTX | Transfecting
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Lipofectamine™ RNAiMAX Transfection Reagent unmatched gene silencing with reduced cytotoxicity for siRNA experiments
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Lipofectamine™ RNAiMAX Transfection Reagent is a proprietary RNAi-specific cationic lipid formulation that offers the highest transfection efficiencies on the widest variety of cell types for siRNA/Stealth™ RNAi gene knockdown experiments:
- Superior transfection efficiency requires lower siRNA/Stealth™ RNAi concentrations and leads to more successful gene knockdown with a minimum of nonspecific effects (Figure 1A)
- Easy optimization due to low cytotoxicity across a 10-fold concentration range of transfection reagent (Figure 1B)
- Versatile approach is compatible with wide-ranging cell types
- Simple and rapid protocol for consistent and reproducible results
RNAiMAX is for use with double stranded RNAi duplexes and not for transfecting RNAi vectors.
Figure 1 - Optimal gene silencing and minimal cytotoxicity with the Lipofectamine™ RNAiMAX transfection reagent.
A. Superior knockdown levels were observed with as little as 0.5 nM Stealth™ RNAi with Lipofectamine™ RNAiMAX Transfection Reagent. Transfection complexes containing 0.3 ml of RNAiMAX were prepared in 48-well plates. A549 cells were added to each well to give final Stealth™ RNAi concentrations of 50 nM to 0.5 nM. Twenty-four hours after addition of cells, p53 knockdown was measured by qRT-PCR with LUXTM primers and was normalized to GAPDH expression. The control duplex in this study was the Stealth™ RNAi Negative Control Medium GC. The percentage of viable cells was measured using the Vybrant™ metabolic activity assay.
B. Minimal cytotoxicity profile over a 10-fold concentration range of Lipofectamine™ RNAiMAX Transfection Reagent. Indicated volumes of transfection reagent were mixed with 10 nM p53 Validated StealthTM RNAi or Stealth™ RNAi Negative Control Medium GC duplexes in 48-well plates. A549 cells were added to each well for a final RNAi concentration of 1 nM. Knockdown of p53 was measured as described in (A). This graph shows that over a 10-fold concentration range of Lipofectamine™ RNAiMAX Transfection Reagent, high levels of gene silencing can be obtained without a dramatic increase in transfection-mediated cytotoxicity.
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Straightforward protocol
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The protocol for using Lipofectamine™ RNAiMAX Transfection Reagent consists of mixing RNAiMAX with siRNA/Stealth™ RNAi, adding cells, incubating, and measuring gene knockdown. The simplicity and speed combined with the superior transfection efficiency make Lipofectamine™ RNAiMAX Transfection Reagent ideal for high-throughput siRNA/Stealth™ RNAi transfections. Transfection conditions can be readily established for automated or robotic systems used in such applications.
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Assessing transfection efficiency
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To qualitatively assess transfection efficiency, we recommend using the BLOCK-iT™ Alexa Fluor® Red Fluorescent Control (Cat. no. 14750-100). Refer to the controls section of RNAi central to learn more on optimizing transfection efficiency.
Note: The BLOCK-iT™ Fluorescent Oligo (Cat. No. 2013) is optimized for use with Lipofectamine™ 2000 Transfection Reagent and does not work well with Lipofectamine™ RNAiMAX Transfection Reagent.
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Lipofectamine™ 2000 Transfection Reagent - the most popular reagent for plasmid and siRNA delivery
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Lipofectamine™ 2000 Transfection Reagent can deliver assorted RNAi reagents, including shRNA and miR RNAi vectors and synthetic molecules such as siRNA, Stealth™ RNAi, and Dicer-generated siRNA pools. Lipofectamine™ 2000 Reagent for RNAi transfection experiments offers many benefits:
- Effective transfection for shRNA and miR RNAi vectors and synthetics (siRNA and Stealth™ RNAi); also works well for co-transfections of synthetics and vectors
- Easy-to-follow protocols; media changes are not required
- Convenient optimization of conditions and transfection efficiency with the BLOCK-iT™ Fluorescent Oligo (both Lipofectamine™ 2000 Transfection Reagent and BLOCK-iT™ Fluorescent Oligo are included in the BLOCK-iT™ Transfection Kit)
- Excellent performance in a wide variety of cell types
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Oligofectamine™ Transfection Reagent - potent internalization of RNA oligonucleotides
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Oligofectamine™ Transfection Reagent was designed for delivery of antisense oligos and is also extremely useful for transfection of siRNA and Stealth™ RNAi into eukaryotic cells. Oligofectamine™ can be used in conjunction with thegreen BLOCK-iT™ Fluorescent Oligo or the BLOCK-iT™ Alexa Fluor® Red Fluorescent Control to optimize and monitor transfection conditions.
In the first published demonstration of siRNA-mediated knockdown in a mammalian cell line, Sayda Elbashir and colleagues reported their use of Oligofectamine™ Transfection Reagent to transfect siRNAs into HeLa cells (1). This work was continued by Jens Harborth et al., who used a similar transfection protocol with Oligofectamine™ to demonstrate the knockdown of 20 additional essential and nonessential target genes. These early studies established the general applicability of using siRNA for RNAi.
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Lipofectamine™ LTX Reagent - maximum gene expression, even in hard-to-transfect and primary cells
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Lipofectamine™ LTX Reagent is a plasmid DNA-specific transfection reagent that provides maximum expression with minimum cytotoxicity. Lipofectamine™ LTX Reagent for RNAi vector transfection experiments offers many benefits:
- High transfection efficiency and significantly lower toxicity levels for a wide range of cell lines
- Significantly improved transfection performance in a number of primary and disease-relevant cell lines
In 90% of the cell lines that we have tested, transfection performance is enhanced by using Lipofectamine™ LTX and PLUS™ Reagent together.
Optimized protocols for over 20 different cell types to save you time
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