RNA Extraction and Real-Time PCR for S. mutans

For RNA extraction, mid-exponential phase (OD_{600 nm} ≅ 0.5) S. mutans cultures were treated with RNAProtect (Qiagen, Inc., Hilden, Germany), and total RNAs were extracted using hot phenol as described previously (Browngardt et al., 2004 (link); Wen et al., 2006 (link), 2011 (link)). To remove residual DNA, RNA samples were treated with RNase-free DNase I (Ambion, Inc., Austin, TX) and total RNA was retrieved with the RNeasy purification kit (Qiagen, Inc.). For real-time PCR analysis, cDNA was synthesized with 1 lg of total RNA using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) by following the procedures recommended by the supplier. Real-time PCR was carried out with a Bio-Rad iCycler using procedures detailed elsewhere (Ahn et al., 2006 (link); Wen et al., 2006 (link)).