Copyright information:Taken from "Evaluation of reference-based two-color methods for measurement of gene expression ratios using spotted cDNA microarrays"BMC Genomics 2006;7():35-35.Published online 24 Feb 2006PMCID:PMC1402275.Copyright © 2006 Peixoto et al; licensee BioMed Central Ltd. 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.