Quantitative RT-PCR Analysis of S. Mutans Biofilm

All RNA was isolated from biofilm (three days). The S. Mutans RNA were isolated and purified by using the Ribopure Kit (Life Technology, Grand Island, NY, USA). A NanoPhotometer P360 (Implen, Westlake Village, CA, USA) was used to quantify the total RNA extracted. Reverse transcription of the RNA into cDNA was carried out by using iScript Advanced cDNA synthesis Kit for RT-qPCR (Biorad, Hercules, CA, USA) according to the manufacturer’s instructions. Real-time PCR was conducted by using iQ SYBR Green Supermix (Biorad, Hercules, CA, USA) (Klein et al., 2010 (link)). The S. Mutans primers for the genes: Glucosyltransferase (gtfB), Glucan-binding protein (gbp), at 10 μM were used. The standard curves were used to transform the critical threshold cycle (Ct) values to the relative number of cDNA molecules. Relative expression was calculated by normalizing each gene of interest to the S. mutans 16S rRNA gene, which is a well-established reference gene (Table 1). PCR amplification was performed by using 20 μL reaction mix per well in a 96 well plate. The reactions were conducted at 95 °C for 3 min, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. After PCR, the melting curve was obtained by incubating the samples at increasing increments of 0.5 °C from 55 to 95 °C (Klein et al., 2009 (link); Koo et al., 2006 (link)).