Optimization of Cell-Based cDNA Microarray Conditions for Gene Functional Studies in HEK293 Cells

Abstract

Since the cell-based cDNA microarray (CBCM) technique has been a useful tool for gain-of-function studies, many investigators have used CBCMs to identify interesting genes. However, this method requires better-established conditions to ensure high reverse transfection efficiency without cross-contamination. Therefore, we optimized CBCM techniques through various means. We determined that Lipofectamine 2000 was the most appropriate transfection reagent by evaluating eight commercialized reagents, and we determined that the most effective concentrations for printing solution constituents were 0.2 M sucrose (to yield a final concentration of 32 mM) and 0.2% gelatin (to yield a final concentration 0.075%). After examining various combinations, we also determined that the best concentrations of cDNA and transfection reagent for optimal reverse transfection efficiency were 1.5 µg/5 µL of cDNA and 5.5 µL of Lipofectamine 2000. Finally, via a time course, we determined that 72 h was the most effective reaction duration for reverse transfection, and we confirmed the stability of cDNA spot activity of CBCMs for various storage periods. In summary, the CBCM conditions that we have identified can provide more effective outcomes for cDNA reverse transfection on microarrays.