September 15, 2006 (Vol. 26, No. 16)
Enhancing Gene Expression Analysis
Transfection is a method used to introduce nucleic acids into mammalian and insect cells. This technique has been used for a wide variety of applications involving protein expression and/or gene analysis. The introduction of a specific gene into cells can be used to help characterize the impact of a particular gene on those cells. This method of research has been important in the areas of drug discovery and gene therapy.
Liposome and non-liposomal reagents are the most common and easy-to-use transfection reagents currently in use. However, all transfection techniques may induce varying levels of cytotoxicity and off-target effects that can mask or alter the cellular response to a gene of interest.
Off-target effects can include unintended up regulation or down regulation of genes within transfected cells. For example, it has been found that various transfection reagents were able to stimulate an interferon-inducible promoter construct in 2C4 cells when transfecting plasmid DNA expressing EGFP. This off-target effect of interferon induction was not observed when transfecting with FuGENE® HD Transfection Reagent.
FuGENE® HD Transfection Reagent is an efficient non-liposomal transfection reagent producing minimal cytotoxicity and high levels of protein expression. In this study, microarray transcriptional profiling experiments were performed in HeLa (ATCC® CCL-2 TM) and MCF-7 cells (ATCC HTB-22 TM) to demonstrate minimal off-target effects when using FuGENE® HD Transfection Reagent compared to a lipid-based transfection reagent (referred to as Reagent L) from another supplier.
Materials and Methods
HeLa and MCF-7 cell lines were plated at 400,000 cells per well in six-well plates. The next day transfections with FuGENE® HD Transfection Reagent or Reagent L were performed according to the manufacturers’ instructions using predetermined optimized ratios of reagent and pM1-MT control plasmid (without insert) or pM1-SEAP, a plasmid expressing secreted alkaline phosphatase (SEAP).
At 48 hours post-transfection, media was removed from the cells and SEAP activity was measured using a chemiluminescent SEAP Reporter Gene Assay Kit (Roche Applied Science; www.roche-applied-science.com) to verify transfection success. SEAP results are found in Figure 1.
Total RNA was isolated following standard protocols. Biotinylated cRNA samples were obtained according to the standard Affymetrix GeneChip® protocol. Hybridization controls (bioB, bioC, bioD, and cre) were added to 15 µg of biotinylated cRNA from the transfected samples. Samples were then hybridized to a Human HG-U133 Plus 2.0 microarray from Affymetrix.
Following hybridization, the chip was washed and stained in an Affymetrix GeneChip Fluidics Station. Stained arrays were scanned with an Affymetrix GeneChip Scanner 3000. Affymetrix GeneChip Operating Software and Quality Reporter were used to quality check and perform data analysis.
Rosetta Biosoftware’s (www.rosettabio.com) Resolver was used to determine ratios with each transfected cell line in the numerator and the cell line-matched control sample in the denominator. Standard filtering criteria for differential expression were applied to each of the eight comparisons: a) ratio p-value <0.001; b) absolute fold-change >1.3-fold; c) log intensity >-0.5. To validate the initial experiment (referred to as experiment 1), the protocol was repeated in a second independent experiment (referred to as experiment 2) approximately 45 days later.
Results and Discussion
The number of genes up or down regulated by the transfection reagent and plasmid complex compared to the untransfected control in two independent experiments (referred to as experiments 1 and 2 with data from experiment 2 indicated in parentheses) is summarized in Figure 2. Black font represents the MCF-7 cell results, and blue font represents the HeLa cell data. Venn diagrams are used to illustrate changes in gene-expression patterns. The overlapping regions indicate the number of transcripts that were altered by both transfection reagents (L and FuGENE® HD Transfection Reagent).
In the first experiment, the total number of genes with altered expression in MCF-7 cells transfected with pM1-MT using FuGENE® HD Transfection Reagent was 197 (Figure 1a). 140 out of those genes also had altered expression when transfected with Reagent L while the other 57 genes were differentially expressed only when MCF-7 cells were transfected with pM1-MT using FuGENE® HD Transfection Reagent. Reagent L had a total of 1,405 genes with altered expression. The gene-expression profiles for the independent second experiment as well as a different cell type (HeLa, blue font) show similar trends.
Although the raw data from the two experiments are different, they may not be statistically significant since there are over 40,000 genes on a single HG U133 Plus 2.0 microarray. The Pearson Correlation Coefficient (value of 1 to be 100% correlated and -1 as 100% inversely correlated) indicates a strong correlation between the two sets of data. All of the data sets have a value of greater than 0.988, with the exception of the HeLa cell sample transfected with pM1-MT using Reagent L. This was expected from this sample, which had low yield of total RNA consistent with the observed cell death in this cultured sample in both experiments.
The gene-expression profiles for pM1-SEAP vector (Figure 1b) transfected into MCF-7 cells (black font) and HeLa cells (blue font) are comparable to those for the control plasmid pM1-MT without insert. They both show a significantly lower number of differentially expressed genes when using FuGENE® HD Transfection Reagent compared to Reagent L.
Even though SEAP activity results (Figure 2) indicated higher levels of SEAP expression from both the HeLa cells and MCF-7 when transfected with FuGENE® HD Transfection Reagent compared to Reagent L, these higher levels of SEAP expression and presumably increased transfection efficiency seen with FuGENE® HD Transfection Reagent-transfected cells do not result in an increase in altered gene expression.
Although the number of genes with altered expression varies depending on the cell type, plasmid, and even the two repeated experiments, there is a significant difference in the impact the two transfection reagents are having on the cells being transfected. Regardless of the variables, FuGENE® HD Transfection Reagent repeatedly demonstrates fewer off-target effects as compared to Reagent L.
The studies presented here indicate significant differences in changes in the gene expression profile of two human cell lines in response to two different transfection reagents. The changes in gene expression, especially in categories such as DNA-dependent transcription, cell cycle, and stress response, are much more pronounced with the lipid-based transfection Reagent L compared to FuGENE® HD Transfection Reagent.
Microarray studies with HEK 293 (ATCC CRL-1573 TM) cells transfected using FuGENE® 6 Transfection Reagent produced similar results to those found here. Reagent L induces a significantly higher number of changes in gene-expression profiles compared to the FuGENE® 6 Transfection Reagent in HEK 293 cells. Researchers performing transfections for the purpose of cellular analysis need to be cognizant of off-target effects associated with various transfection methods, which may lead to misinterpretation of resulting data.
FuGENE® HD Transfection Reagent has been shown to have minimal off-target effects and is an ideal transfection reagent for these applications.
Jay Wang is a member of the R&D team at Roche Diagnostics. Web: www.roche-applied science.com. com. E-mail: [email protected]. For a more detailed explanation of the work discussed here, please see the upcoming article in Biochemica, Roche Applied Science’s quarterly newsletter (www.roche-applied science.com/publications/biochemica.htm).