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Mining the “meaningful” clues from vast amount of expression profiling data remains to be challenge for biologists. After all the statistical tests, biologists often struggle deciding how to do next with a large list of genes without any obvious theme of mechanism, partly because most statistical analyses do not incorporate understanding of biological systems before hand. Here, we developed a novel method of “gene –pair difference within a sample” to identify phenotype-defining gene signatures, based on the hypothesis that a biological state is governed by the relative difference among different biological processes. For gene expression, it is relative difference among the genes within a sample (an individual, cell, etc), the highest frequency of occurrences a gene contributing to the within sample difference underline the contributions of genes in defining the biological states. We tested the method on three datasets, and identified the most important gene-pairs to drive the phenotypic differences.