PROTOCOL
16S rRNA-based, reverse-capture oligonucleotide probes (typically 18 to 20 bases) are printed on aldehyde-coated glass slides (Figure 1). 16S rRNA genes are PCR amplified from DNA extracts using 16S rRNA universal forward and reverse primers and labeled via incorporation of Cy3-dCTP in a second nested PCR. The labeled 16S amplicons are hybridized overnight to probes on the slides. After washing, the microarray slides are scanned using an Axon 4000B scanner and crude data is extracted using GenePix Pro software. Data are normalized by comparing individual signal intensities to the average of signals from universal probes.
Figure 1: Microarray slide. (A) Each array is comprised of 4 sections, shown vertically. Each slide has 5 sets of arrays in replicate. (B) A single 8X15 section of an array is magnified, illustrating oligonucleotide probes spotted on the slide.
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