Oligoanalyzer software

Genomic DNA (gDNA) was isolated from cultured bacterial cells using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. Oligonucleotides used in this study (Table 1) for the amplification of cariogenic bacteria (S. mutans, Lactobacillus spp., and Actinomyces spp.), periodontal bacteria (A. actinomycetemcomitans, P. gingivalis, T. forsythia, T. denticola, P. micra, P. intermedia, and F. nucleatum) and for the determination of total bacteria (TB) content in the samples were based on published sequences or designed de novo using Primer3 (v. 0.4.0) [16 (link)] and OligoAnalyzer software (Integrated DNA Technologies, Inc., Coralville, IA, USA) [17 (link)] (Table 1).
Specific forward and reverse oligonucleotides (Table 1) were used to amplify 100–232 bp fragments from the target regions for each of the bacterial species (Table 1), and the amplified DNA fragments were cloned into the cloning vector pGEM^®-TEasy (Promega, Madison, WI, USA). The correctness of these cloned DNA fragments was verified by sequencing using T7FW and M13R primers (data not shown).

Differentially expressed genes were selected for quantitative PCR (qPCR) analysis to confirm the microarray data. Specific primers were designed with the OligoAnalyzer software (Integrated DNA Technologies) (see Table S3 in the supplemental material). Amplified aRNA samples (5 µg) were reverse transcribed using the M-MuLV (Moloney murine leukemia virus) Taq reverse transcriptase (RT)-PCR kit (New England Biolabs, Ipswich, MA), following the manufacturer’s instructions. qPCR consisted of a reaction mixture with 10 µl SYBR green master mix (BioRad, Hercules, CA), 10 µl containing forward and reverse primers (150 nM each), and 10 ng template. Reactions were performed in triplicate under the following conditions: 5 min of denaturation at 95°C, followed by 40 cycles of denaturation at 95°C for 30 s, primer annealing at 60°C for 30 s, and extension at 72°C for 30 s. Melting curve analysis was used to confirm template specificity and sample purity, and results were analyzed using the cycle threshold (ΔΔC_T) method (47 (link)). The expression levels of two housekeeping genes, gyrB and rpoA, were used as controls. Statistical significance was calculated with the Student t test.