Schematic representation of the experimental design. (A) Human cDNA inserts that were cloned into pUC18 vector were amplified by PCR with vector universal primers in such a way that each PCR-amplified cDNA probe spotted on the array contained a common 5'-end sequence derived from the vector. The RefOligo consisted of a synthetic Cy3-labeled 27-mer oligonucleotide (marked with green asterisks) that is complementary to the 5'-end vector sequence present in each cDNA probe. This RefOligo common reference was co-hybridized to the arrays along with Cy5-labeled tissue-derived targets (marked with red asterisks). (B) Overview of the experimental design including direct and indirect hybridizations. Filled squares represent fluorescent targets derived either from test RNAs (B, Breast; K, Kidney; P, Prostate), or from two types of external references, RefPool (Rp) or RefOligo (Ro). Arrows indicate co-hybridized target RNAs -- arrow head indicate the target labeled with Cy3, whereas the arrow tail indicate the target labeled with Cy5. The number of replicate hybridizations performed with each paired sample is indicated in the small circles.