Figure 6

Hybridization signal intensity on the array obtained with different amounts of synthetic polyA-RNA spiked in. Synthetic polyA-RNA species were generated using sequences of bacteriophage λ. An oligo (A)₃₀ sequence at each 3'end was incorporated in the course of a PCR reaction. Templates for in vitro transcription with T7 RNA polymerase were engineered by cloning in a vector containing a T7 promotor and digesting with HIND III. The transcripts were purified with Oligotex mRNA Minikit (Qiagen, Hilden, Germany) to get pure polyA-transcripts. A) Different amounts of three synthetic polyA-transcripts (B: light blue; H: margenta; J: black) were spiked in as exogenous controls for labelling, hybridization and detection. The hybridization signals determined after 10 min incubation with the HRP substrate are presented versus the amounts of controls spiked in. B) Data analysis at 2.5, 5 and 10 min, respectively, after incubation with the HRP substrate, shown for three different synthetic RNA concentrations (black: 3.2E-9 nmol of sequence J, light blue: 6.48E-9 nmol of sequence B, margenta: 1.14E-8 nmol of sequence H). This way, the dynamic range of array analysis can be expanded to some extent.