Transcriptional Analysis of Streptococcus mutans Biofilm Development

RNA was extracted and purified from biofilms at distinct stages of microcolonies development (21, 24, 30, 31, and 34 h) using standard protocols [22] (link). The developmental stages of S. mutans biofilms were characterized previously [21] (link), [23] (link), which varies from initial microcolonies assembly across the apatite surface (at 20 h) to vertical growth and merging process followed by further increase in size and thickness (from 20 to 30 h and beyond). All purified RNAs presented RNA integrity number (RIN) ≥8.5 (Agilent 2100 electrophoresis bioanalyzer, Agilent Technologies, Santa Clara, CA, USA). The reverse transcriptase PCR, and quantitative amplification conditions were similar to those described previously [20] (link). The primers were designed using Beacon Designer 2.0 software (Premier Biosoft International, Palo Alto, CA) (see Table 1).
Briefly, cDNAs were synthesized using the BioRad iScript cDNA synthesis kit (Bio-Rad Laboratories, Inc., CA). To check for DNA contamination, purified total RNA without reverse transcriptase served as a negative control. The resulting cDNA and negative controls were amplified by a MyiQ qPCR detection system with iQ SYBR Green supermix (Bio-Rad Laboratories, Inc., CA, USA) and specific primers. A standard curve was plotted for each primer set, as described elsewhere [20] (link). The standard curves were used to transform the quantification cycle (Cq) values to the relative number of cDNA molecules. Relative expression was calculated by normalizing each gene of interest to the reference gene 16S rRNA [20] (link).