The single-stranded large circular (LC)-sense DNA were utilized as probes for DNA chip experiments. The microarray experiment using LC-sense DNA probes found differentially expressed genes in A549 cells as compared to WI38VA13 cells, and microarray data were well-correlated with data acquired from quantitative real-time RT-PCR. A 5K LC-sense DNA microarray was prepared, and the repeated experiments and dye swap test showed consistent expression patterns. Subsequent functional analysis using LC-antisense library of overexpressed genes identified several genes involved in A549 cell growth. These experiments demonstrated proper feature of LC-sense molecules as probe DNA for microarray and the potential utility of the combination of LC-sense microarray and antisense libraries for an effective functional validation of genes.