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Application Note
Nature Methods - 3, (2006)
Published online: 20 April 2006; | doi:10.1038/nmeth879

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FuGENE® 6 Transfection Reagent: minimizing reagent-dependent side effects as analyzed by gene-expression profiling and cytotoxicity assays

Transfection protocols and the reagents used to deliver nucleic acids into cells can affect cell physiology, leading to misinterpretation of results. Using microarray analysis and cell viability assays, we evaluated different transfection reagents to determine their effects on gene expression profiles. Results show that FuGENE 6 Transfection Reagent exhibits very low cytotoxicity, delivers high transfection efficiency and generates few nonspecific side effects with minimal alteration in gene expression.
FuGENE 6 Transfection Reagent is a nonliposomal, multicomponent reagent proven to efficiently transfect more than 700 cell types. It can be used in the presence or absence of serum, and its minimal cytotoxicity eliminates the need to change medium after transfection. Thousands of peer-reviewed publications have proven FuGENE 6 Transfection Reagent´s ability to transfect many eukaryotic cell types with higher efficiency and greater cell survival than previously attainable. Compared to other transfection methods, FuGENE 6 Transfection Reagent gives more consistent results, saves time and effort, and has minimal effect on cell physiology.

Delivery of nucleic acids into cells using liposomal or multicomponent transfection reagents is a well-established method for transfection of mammalian cell lines. This approach has been widely used (for example, for the overexpression of proteins with expression plasmids or for specific gene knockdown experiments using siRNA). Many transfection reagents, however, are known to exhibit substantial toxic side effects, as reflected by cell viability assays. Such effects on cell physiology are often underestimated or neglected; moreover, side effects may lead to misinterpretation of results. Therefore, evaluation and selection of a suitable transfection reagent is crucial to achieve valuable results with minimal side effects.

Microarray analysis allows monitoring of gene expression of thousands of genes in parallel. This application allows functional genomic analysis of transcription patterns in different tissues, in different stages of development or in various diseases. Using microarray technology, changes in expression profiles of cells treated with toxins or drugs can also be explained. Furthermore, nonspecific side effects can be detected with microarray technology. These could include off-target effects induced by the transfection procedure rather than by the specific gene knockdown, or the activation of the interferon system in a series of siRNA transfection experiments1, 2. The question remains open as to what extent transfection procedures in general, or certain transfection reagents, lead to changes in the expression profile.

To analyze the influence of different transfection reagents on the expression profile of transfected cells, we tested FuGENE 6 Transfection Reagent in comparison to reagent A, a widely used transfection reagent from a different supplier. We carried out transfections of HEK 293 cells with three different plasmids. (i) To determine the impact of the transfection reagent or procedure, we transfected a plasmid without an insert. (ii) To determine the influence of overexpression of a secreted protein, we transfected a plasmid coding for the secreted human placental alkaline phosphatase (SEAP). (iii) As an example for a cytoplasmic protein, we transfected a plasmid coding for enhanced green fluorescent protein (EGFP). EGFP is known to induce apoptosis3.

To investigate whether the transfection reagent causes side effects, we compared gene expression (using human GeneChip HG U133 Plus 2.0) after transfection of HEK 293 cells with either the FuGENE 6 Transfection Reagent or reagent A. We analyzed the expression profile of cells transfected with a plasmid without insert (pM1-MT) compared with one coding for the secreted protein SEAP (pM1-SEAP). We analyzed the expression profile of cells transfected with a plasmid coding for the intracellular protein EGFP using FuGENE 6 Transfection Reagent only.

Expression patterns
We analyzed microarray data sets with the RACE-A software (Roche Affymetrix Chip Experiment Analysis). For each plasmid, we performed three transfections and three microarray hybridizations. We grouped the data from these three individual results and compared them with the microarray pattern from an untransfected control. Our results showed that transfection influences the expression pattern of HEK 293 cells, depending on the transfection reagent and the transfected plasmid. The transfection using FuGENE 6 Transfection Reagent had an effect on 30 different genes, in contrast to that using reagent A, which affected 2,061 genes with a change in expression level of at least two fold. By transfecting a plasmid without insert, expression levels of 12 genes were affected by both transfection reagents, indicating a specific pattern for the transfection procedure with this plasmid (Fig. 1a).

Figure 1. Alterations in gene expression after transfection.
Figure 1 thumbnail

(a,b) The changes in expressed genes were compared between transfections with FuGENE 6 Transfection Reagent and reagent A for the plasmid without insert (a) and the plasmid coding for SEAP (b). (c,d) The number of affected genes after transfection with FuGENE 6 Transfection Reagent (c) was compared with those after transfection using reagent A (d). Overlapping areas represent genes with changed expression levels for all the plasmids (c and d) or by both transfection reagents (a and b).



Full FigureFull Figure and legend (55K)
After transfecting a plasmid coding for SEAP, the expression of 72 genes was altered using FuGENE 6 Transfection Reagent, compared with 2,743 affected genes using reagent A. In this experimental setup, 50 genes showed changed expression with both reagents. These genes might be specific for SEAP expression via the plasmid pM1-SEAP (Fig. 1b). Transfection of three different plasmids using FuGENE 6 Transfection Reagent affects only a small number of genes, suggesting that FuGENE 6 Transfection Reagent minimizes reagent-dependent alterations (Fig. 1c). In contrast, reagent A causes a massive change in the expression pattern of a total of 1,617 genes with both of the transfected plasmids (Fig. 1d).

Cell viability
Furthermore, the changes in the expression profile correlate to the results of viability assays. We analyzed cell viability using the Cell Proliferation Reagent WST-1 (Roche Applied Science) according to the instructions. We found that the transfection experiment displaying least influence on the expression pattern shows the highest viability of cells (FuGENE 6 Transfection Reagent with plasmid without insert). In contrast, the transfection using reagent A and the SEAP expression plasmid shows the highest alteration of expression levels and lowest cell viability (data not shown).

Influence on cell physiology–related gene expression
To determine the relevance of genes with altered expression levels, we analyzed genes coding for proteins that have a crucial role in cell physiology. Cells transfected with FuGENE 6 Transfection Reagent and the plasmid without insert show dysregulation of only a very small number of genes involved in cell signaling and cell cycle, and no change in the regulation of genes involved in apoptosis, stress response or immune response (Table 1). Transfection with pM1-SEAP using FuGENE 6 Transfection Reagent influences a slightly higher number of genes with these functions. As expected, transfection of a plasmid coding for intracellular EGFP affected more genes involved in crucial cellular functions—most likely because of its apoptotic properties. Dysregulation of cell physiology—related genes increased exceedingly after transfection using reagent A.

Table 1. Changes in gene expression profiles caused by different transfection reagents
Table 1 thumbnail

Full TableFull Table
Transfection efficiency
In contrast to the differences observed concerning the viability of transfected cells and the alterations in gene expression profiling, both transfection reagents show similar transfection efficiency (Fig. 2).

Figure 2. Efficiency of transfection using FuGENE 6 Transfection Reagent and reagent A with two different plasmids coding for SEAP and EGFP.
Figure 2 thumbnail

Amounts of recombinant proteins were determined 48 h after transfection and related to total protein. For each plasmid, the highest efficiency was set to 100%.



Full FigureFull Figure and legend (20K)
Conclusion
The transfection reagents investigated here have very different impacts on gene expression patterns in HEK 293 cells. Reagent A results in a dramatically changed expression profile. In contrast, FuGENE 6 Transfection Reagent causes only minimal alteration of gene expression. Furthermore, expression levels of only a few genes with a crucial role in cell physiology are affected. Besides the marginal changes in expression levels, FuGENE 6 Transfection Reagent shows very little cytotoxicity. These features make FuGENE 6 Transfection Reagent a superior tool for transfection experiments in general. It is especially suited for functional studies, yielding valuable results with minimal side effects.

FuGENE is a registered trademark of Fugent, LLC, USA. For details of the experiments described above, see Biochemica 4, 9–11 (2004), available online (http://www.roche-applied-science.com/publications/biochemica.htm).

This article was submitted to Nature Methods by a commercial organization and has not been peer reviewed. Nature Methods takes no responsibility for the accuracy or otherwise of the information provided.

Published online: 20 April 2006.

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REFERENCES
  1. Jackson, A.L. et al. Nat. Biotechnol. 21, 635–637 (2003). | Article | PubMed | ISI | ChemPort |
  2. Sledz, C.A. et al. Nat. Cell Biol. 5, 834–839 (2003). | Article | PubMed | ISI | ChemPort |
  3. Liu, H.S. et al. Biochem. Biophys. Res. Commun. 260, 712–717 (1999). | Article | PubMed | ISI | ChemPort |
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Authors

Vivien Nagy & Manfred Watzele

Roche Applied Science, Nonnenwald 2 D-82372 Penzberg, Germany.

Correspondence should be addressed to Manfred Watzele manfred.watzele@roche.com

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