Introduction

The insertion of exogenous DNA or RNA into a cell through the process of transfection is an important, well-established tool for molecular and cellular biologists. The application of transfection as a research tool includes the production of the protein encoded by an exogenous DNA that is either transiently maintained in the nucleus or has been integrated into the genome of the host cell, resulting in the production of a stable cell line. Other uses include: (i) introduction of short interfering RNA (siRNA) or short hairpin RNA (shRNA) to examine the impact of the decreased expression of a particular gene, (ii) studying the effects of a constitutively active or dominant negative enzyme construct, and (iii) identifying the subcellular localization of a protein by using a construct encoding the gene of interest fused to the coding region for a marker such as green fluorescent protein (GFP).

The two most common approaches for transfecting mammalian and insect cells involve either the use of chemical reagents (usually, liposome-based) or the application of an electrical current (electroporation), which is typically used for cells grown in suspension or that are difficult to transfect using chemical reagents. Unfortunately, many transfection reagents and electroporation have considerable cytotoxic effects on the cells. Furthermore, it has long been recognized that different transfection reagents cause different levels of cytotoxicity in different cell lines. These effects are often directly attributable to the transfection reagents themselves and not the nucleic acid of interest that is being introduced to the cell.

Additionally, published literature suggests that there are a variety of negative effects resulting from cryptic or ‘poison’ sequences within the plasmid vector itself (1). This raises the question of what other effects might be a direct result of the transfection event itself or other sites in the vector and not due to the presence of the exogenous RNA or DNA selected for study. Clearly, understanding these off-target effects is important to the proper interpretation of results generated using transfected cells. Initial studies have been done that indicate these changes can be identified at the level of transcription (www.roche-applied-science.com/PROD_INF/BIOCHEMI/no4_06/pdf/22.pdf).

For these reasons, we chose the common model cell line MCF7 to identify the effects of different transfection reagents. To pinpoint potential off-target transcriptional effects, we performed microarray-based gene expression profiling assays on cells that had been transfected with four different commercially available transfection reagents at three different time points. The microarray data was used to determine genes and biological processes that were consistently modified in the presence of only the gene of interest, or as a result of exposure to a given transfection reagent. Our findings indicate that transfection-mediated transcriptional effects need to be fully appreciated by researchers within their own model systems. This information might be used to prevent the misinterpretation of results generated when studying a specific gene of interest.

Materials and methods

Transfection

MCF7 (ATCC HTB-22) cells were obtained directly from ATCC (Manassas, VA, USA) and maintained under stringent standard operating procedures to prevent cross-contamination. Cells were plated 1 day before transfection at a density of 400,000 cells/well in a 2-mL volume in 6-well plates. MCF7 cells were grown in EMEM (Cat. no. 11090081; Invitrogen, Casrlsbad, CA, USA) supplemented with 10% FBS (Cat. no. SH30070.03; Hyclone Laboratories, Logan, UT, USA), glutamine (Cat. no. 25030081; Invitrogen), non-essential amino acids (Cat. no. 11140050; Invitrogen), sodium pyruvate (Cat. no. 11360070; Invitrogen), and human recombinant insulin (Cat. no. 11376497001; Roche Applied Science, Indianapolis, IN, USA). Transfections with all reagents were performed according to the manufacturer's protocols which included performing optimization experiments to determine the most ideal transfection conditions for each reagent. In instances in which several parameters were found to be optimal for a given transfection reagent (with regard to the ratio of the volume of transfection reagent and the mass of exogenous DNA), different ratios were tested in some experiments. Table 1 lists the reagents and amounts used in the experiments performed for microarray analysis. Reagents were tested in two to four separate experiments for the 48-h time point; shorter time points were tested in fewer experiments. Four to twelve independent transfections were done in each experiment for use in microarray analysis. Two different vectors were used within the context of this project. The pM1-SEAP is a vector expressing secreted alkaline phosphatase (SEAP) and was used in all four experiments. The pM1-MT is a control vector containing an identical backbone to pM1-SEAP, but without the SEAP insert, which was used in two experiments. Vectors were diluted in OPTIMEM I Reduced Serum Medium (Invitrogen) or buffer supplied with reagent. SEAP was selected for these studies as it is secreted and has minimal effects on the biological processes within a cell (http://www.roche-applied-science.com/PROD_INF/BIOCHEMI/no4_04/PDF/p09.pdf).