Biofilm preparation. Hydroxyapatite discs (2.93 cm2) were coated with amylase-active sterile clarified human saliva (sHA). Biofilms of S. mutans UA159 were formed on sHA discs placed in a vertical position in batch cultures at 37oC in 5% CO2. The biofilms were grown in buffered tryptone yeast-extract broth, pH 7.0, containing specific concentrations of sucrose and/or starch (soluble starch – 80% amylopectin and 20% amylose; Sigma Chemical Company, St Louis, MO). The concentrations of 0.5% sucrose (suc), 0.5% sucrose + 1% starch (st) and 1% sucrose were used.
Extracted molecule
total RNA
Extraction protocol
The biofilms were removed from HA disc in sufficient volume of RNALater, according to manufacture instructions, and stored at 4oC, overnight. Next day, the biofilm samples were washed with cold 1X PBS and sonicated at 7 watts for 30 sec (2 sonication-washings). The supernatant was discarded and biofilm pellets were homogenized and RNA extracted by repeated acid-phenol: chloroform extractions, and the nucleic acid was precipitated with 1 volume ice-cold isopropanol and one-tenth volume 3M sodium acetate (pH 5) at –20°C overnight. RNA pellets were resuspended in 50 μL nuclease-free H2O, and treated with DNase I to remove contaminant genomic DNA. Two types of commercially available DNase I treatments were used: (i) on-column DNase I (Qiagen RNeasy Mini Kit, Qiagen Sciences, Germantown, MD, USA) and (ii) protein-engineered DNase I in solution (Turbo DNase, Ambion). An aliquot of the extracted RNA was treated first using Qiagen’s RNeasy on-column DNase I (Q, 2.7 U DNase I/10 μg RNA) and then with Ambion’s Turbo DNase I (T, 2 U Turbo DNase I/10 μg RNA), followed by Qiagen RNeasy MinElute (for DNase I removal) according to the manufacturer’s protocols with the exception of Qiagen’s on-column DNase I. The RNeasy on-column DNase I protocol was modified by adding a washing step with 350 μl of RLT buffer containing 36% ethanol prior to column washing with RW1 buffer (RLT and RW1 buffers are proprietary solutions of Qiagen Sciences). RNA concentrations were determined using a Nanodrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA).The quality of the purified RNA was examined by an Agilent 2100 electrophoresis bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) to verify RNA integrity.
Label
Cy3
Label protocol
Once RNA was purified as described above, cDNA was generated from 2ug RNA per sample as described by the protocol available from the Pathogen Functional Genomics Research Center (PFGRC) using Invitrogen Superscript III Reverse Transcriptase (Invitrogen, Gaithersburg, MD) to increase cDNA yields. Purified cDNAs were coupled to Cy3 (experimental samples) or Cy5 (reference cDNA).
Cells were grown in Brain-Heart medium to OD600 of 0.5 in a 5% CO2 incubator at 37°C.
Extracted molecule
total RNA
Extraction protocol
The biofilms were removed from HA disc in sufficient volume of RNALater, according to manufacture instructions, and stored at 4oC, overnight. Next day, the biofilm samples were washed with cold 1X PBS and sonicated at 7 watts for 30 sec (2 sonication-washings). The supernatant was discarded and biofilm pellets were homogenized and RNA extracted by repeated acid-phenol: chloroform extractions, and the nucleic acid was precipitated with 1 volume ice-cold isopropanol and one-tenth volume 3M sodium acetate (pH 5) at –20°C overnight. RNA pellets were resuspended in 50 μL nuclease-free H2O, and treated with DNase I to remove contaminant genomic DNA. Two types of commercially available DNase I treatments were used: (i) on-column DNase I (Qiagen RNeasy Mini Kit, Qiagen Sciences, Germantown, MD, USA) and (ii) protein-engineered DNase I in solution (Turbo DNase, Ambion). An aliquot of the extracted RNA was treated first using Qiagen’s RNeasy on-column DNase I (Q, 2.7 U DNase I/10 μg RNA) and then with Ambion’s Turbo DNase I (T, 2 U Turbo DNase I/10 μg RNA), followed by Qiagen RNeasy MinElute (for DNase I removal) according to the manufacturer’s protocols with the exception of Qiagen’s on-column DNase I. The RNeasy on-column DNase I protocol was modified by adding a washing step with 350 μl of RLT buffer containing 36% ethanol prior to column washing with RW1 buffer (RLT and RW1 buffers are proprietary solutions of Qiagen Sciences). RNA concentrations were determined using a Nanodrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA).The quality of the purified RNA was examined by an Agilent 2100 electrophoresis bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) to verify RNA integrity.
Label
Cy5
Label protocol
Once RNA was purified as described above, cDNA was generated from 2ug RNA per sample as described by the protocol available from the Pathogen Functional Genomics Research Center (PFGRC) using Invitrogen Superscript III Reverse Transcriptase (Invitrogen, Gaithersburg, MD) to increase cDNA yields. Purified cDNAs were coupled to Cy3 (experimental samples) or Cy5 (reference cDNA).
Hybridization protocol
The slides were hybridized to a mixture containing equal amounts of test and reference cDNA for 16 h at 42°C in a MAUI hybridization chamber (BioMicro Systems). Hybridized slides were washed and scanned using a GenePix scanner (Axon Instruments).
Scan protocol
Hybridized slides were washed and scanned using a GenePix scanner (Axon Instruments, Inc., Union City, CA).
Data processing
Data were analyzed using software available at the J. Craig Venter Institute PFGRC website. Single-channel images of the slides were loaded into Spotfinder and overlaid. A spot grid was created according to PFGRC specifications and manually adjusted to fit all spots. The Microarray Data Analysis System (MIDAS) software was used to normalize the spot intensity data using LOWESS and standard deviation regularization with default setting. MIDAS also utilized In-Slide Replicate Analysis to average the intensities of the spots that were replicated on the microarray slide. Statistical analysis was carried out with BRB array tools (http://linus.nci.nih.gov/BRB-ArrayTools.html) with a cutoff P value of 0.001.
