Extraction and purification of total RNA from Streptococcus mutans biofilms.
RNA isolation from Streptococcus mutans within biofilms is challenging because of the presence of extracellular polysaccharide matrix that interferes with RNA extraction procedures. In an effort to solve this difficult problem, we examined several protocols to extract and purify RNA from S. mutans biofilms. A combination of sonication (three times using a 30-s pulse at 7 W) with washing in phosphate-buffered saline removed most of the extracellular polysaccharides from the biofilms and provided the highest RNA yield. Further homogenization-mechanical cells disruption in NAES buffer (50 mM sodium acetate buffer, 10 mM EDTA, and 1% SDS, pH 5.0) and acid phenol/chloroform yielded 547.2+/-23.4 microg RNA/100 mg of biofilm dry weight. An additional acid phenol/chloroform extraction further improved the purification of RNA without significantly affecting the RNA yield. The combination of DNase I in silica gel-based column and recombinant DNase I in solution effectively removed the genomic DNA as determined by real-time quantitative reverse transcriptase PCR (RT-PCR), resulting in 92.0+/-0.6 microg of purified RNA per 100 mg of biofilm dry weight. The complementary DNAs generated from the purified RNA sample were efficiently amplified using gtfB S. mutans-specific primers. The results demonstrated a method that yields high-quality RNA from biofilms of S. mutans in sufficient quantity for real-time RT-PCR analyses, and our data have relevance for isolation of RNA from other biofilm-forming microorganisms.
Cury
Jaime A
JA
Faculty of Dentistry of Piracicaba, State University of Campinas, Piracicaba, São Paulo, Brazil.
Koo
Hyun
H
D018441:Biofilms; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012329:RNA, Bacterial / Q000302:isolation & purification*; D013295:Streptococcus mutans / Q000235:genetics* / Q000254:growth & development*
D012329:RNA, Bacterial