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Gene expression profiling studies are commonly used to study signaling pathways and their impact on transcriptional regulation in plants. In some cases, a profiling study results in expression profiles in which most genes exhibit a small number of differentially expressed states among a large number of samples. In such instances, a pooling approach would help improve the efficiency of the profiling effort by employing fewer microarray chips and ensuring more robust measurement of transcript levels. Smart pooling involves pooling of mRNA samples in an information-efficient manner such that each sample is tested multiple times but always in pools with other samples. The resulting pooled measurements are then decoded to recover the expression profile of all samples in the study. In this protocol, we describe in detail the process of designing smart pooling experiments and decoding their results, which have been used for studying signaling in Arabidopsis root development. Heuristics are provided to select the design parameters that would ensure successful execution of smart pooling.