In our system, an equimolar oligo reference is defined as an equimolar mixture of oligos that hybridize perfectly and uniquely to the oligo features on an array, e.g. the reverse complementary sequences corresponding to the features on an oligo array. Because the synthesis of a large number of 70-mers was not economically feasible, we used a spiked oligo experiment as a proof of concept study (diagrammed below). Oligos complementary to spots with low expression levels in a specific cDNA sample were labeled and spiked into a hybridization reaction containing that cDNA sample. Cy3-labeled oligos were added as an equimolar mixture, while Cy5-labeled oligos were added as a 5-fold dilution series, simulating the equimolar oligo reference and cDNA transcripts, respectively.