Comparative Microarray Gene Expression Analysis

Both a PCR gene product microarray and a 70-mer oligonucleotide-based microarray (tuberculosis oligonucleotide set; QIAGEN) were used. Labeled cDNA was prepared as follows: 2 μg total RNA and 4.4 μg of random oligonucleotide hexamers were incubated for 2 min at 98°C, cooled on ice, combined with Stratascript RTase buffer, 0.5 mM dA,G,CTP, 0.02 mM dTTP, 1.5 nmol Cy3 or Cy5-dUTP (Amersham Biosciences), and 1.8 μl Stratascript RTase (Stratagene) in a total volume of 25 μl, and incubated for 10 min at 25°C and 90 min at 42°C. cDNA was purified by microcon-10 (Amicon) filtration. 10 μl of hybridization solution (labeled cDNA, 5 μg tRNA, 3.8× SSC, 0.27% SDS) was sealed under a coverslip with rubber cement and hybridized overnight at 65°C for the DNA microarray. Oligonucleotide microarrays were first prehybridized for 1 h in 5× SSC, 1% BSA, and 0.1% SDS and washed with H₂O and isopropanol. After the prehybridization, 10 μl of hybridization solution (labeled cDNA, 5 μg tRNA, 2× SSC, 25% formamide, and 0.1% SDS) was hybridized overnight at 54°C.