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.