A detailed description of the Choe et al.  experiment. Individual PCR products (a) were pooled together (b) and converted to labeled cRNA (c). Note that all mixing and labeling within each pool was performed at this stage, before splitting the pools into C and S samples. Therefore, relative concentrations of individual cRNA species are identical for all cRNAs in a given pool. (d) The labeled pools were then divided into the C and S samples. Poly(C) RNA (20 μg) was added to the C sample at this step to equalize the amount of nucleic acid present in each hybridization. (e) Each sample contained enough labeled cRNA for three hybridizations. Relative concentrations for each pool are shown in (f). Note that the 1× ("null gene") pools 13-19 were combined together at step (b), before labeling at step (c), creating a single 1× pool before labeling and splitting. The '1×' concentration of RNA used for this pool was approximately 6× greater than the 1× concentrations of the other pools to reflect the greater number of individual RNAs (that is, so that the 1× concentrations of all RNAs were approximately equal).