Microarray Hybridization, Washing, and Scanning

The preparation of chips for hybridization, target hybridization, washing, and scanning were performed by the procedure described as follows (LC Science, Houston, TX, United States). Briefly, the mixture of 25 μl of PCR amplification products (200 ng/μl) and 25 μl of hybridization buffer was added on the in situ-synthesis gene chip. Subsequently, hybridization for 18 h at 40°C, washing at 40°C, and staining were performed in a fluidics station at LC Science. The hybridization buffer (1 ml) was flushed through the chip at 500 μl/min for 20 min. The array chips were scanned using a GenPix 4000B scanner (Molecular Devices/Axon, Sunnyvale, CA, United States). The scanning pixel size was 10 mm. The Cy3 signals were collected using 532-nm channels (Zhu et al., 2007).