The procedure is a modification of the previously reported 'Round ABC' protocol (25) . Briefly, the RT-PCR reaction was assembled at 48C in a PCR workstation and transferred to a thermocycler with the block preheated to 508C. The initial reverse transcription was performed at 508C for 30 min and followed by 958C for 15 min to activate HotStarTaq DNA polymerase as well as to inactivate the reverse transcriptases (Round A). The PCR conditions were 35 cycles at 928C for 30 s, 558C for 30 s and 688C for 1.5 min with a final extension step at 688C for 5 min (Round B). One microliter of unpurified product was subsequently used as a template for another 20 cycles of amplification to label the amplicons via a previously described 'Round C' PCR protocol (948C for 30 s, 408C for 30 s, 508C for 30 s and 728C for 1 min) with primer B and a 4:1 mixture of aminoallyl dUTP (Ambion, Austin, TX, USA) and dTTP for probe labeling (25) . The labeled amplicons were purified with DNA Clean-up and Concentrator-5 columns (Zymo Research, Orange, CA, USA), eluted in 9 ml of sodium bicarbonate (pH 9.0) and coupled with 1 ml of DMSO dissolved Cy3 NHS esters (GE Healthcare, Piscataway, NJ, USA) for 30-60 min. The Cy3-labeled amplicons were purified with DNA Clean-up and Concentrator-5 columns and eluted with 10 ml of 10 mM Tris-HCl (pH 8.0). Then, the Cy3-labeled amplicons were diluted in water and combined with 3.6 ml of 20Â SSC, 0.5 ml of HEPES (pH 7.0) and finally 0.5 ml of 10% SDS to reach final volume of 25 ml. The mixed solution was heated for 2 min at 958C, cooled to room temperature and hybridized to the exon mapping arrays at 638C overnight essentially as previously described (25) (26) (27) . The hybridized arrays were washed and scanned with a GenePix 4000B scanner (Molecular Device, Sunnyvale, CA, USA) and analyzed by GenePix Pro 6.0 software.