Comprehensive transcriptome analysis of Porphyromonas gingivalis using high-density tiling microarrays

High-density tiling microarrays contain hundreds of thousands of oligonucleotide probes enough to cover every nucleotide of a bacterial genomic sequence. These probes, when used in hybridization experiments, provide unbiased and comprehensive information for the transcriptional activities of the organism. Objective: To define the complete transcriptome for Porphyromonas gingivalis W83 using the high-density tiling microarrays. The result can provide experimental evidences for the computer-predicted genes and can discover novel ones that were not predicted before. Methods: The probes were designed by a program to dynamically select a set of oligonucleotide sequences that are unique, thermodynamically optimal, and cover the entire genome in both forward and reverse-complement directions with an average density of one probe per 12 bases. A total of 385,000 50-base oligonucleotides were designed and were printed on photolithography-based microarrays manufactured by NimbleGen. RNA samples were extracted from P. gingivalis cultures grown under laboratory conditions. Single-strand cDNAs were synthesized from the RNA, and then labeled with biotinylated ddATP by terminal transferase end-label reactions. The labeled cDNAs were hybridized with the probes on the arrays and the result scanned after staining with fluorolink cy3-streptavidin. The intensity profiles were normalized with DNA reference arrays and RNA transcription units identified by the BioConductor tilingArray package. Results: We have confirmed the expression of many hypothetical genes that were predicted by computational methods. We have also identified many novel transcripts in both intergenic and anti-sense regions. The expression of selected novel RNAs were verified by reverse transcription PCR. Conclusion: This study provides experimental validation for genomic annotations and discovered novel coding and non-coding RNAs that may play important roles in disease mechanisms for P. gingivalis. The identification of novel RNAs will foster new hypothesis-driven studies to investigate their functions. Supported by NIH grant NIDCR K22 DE14742 and by The Faculty of Dentistry, University of Oslo.