Briefly, 50-ml cultures that were intended for use in microarray experiments were grown under the desired conditions and harvested by centrifugation at 4°C. Pelleted cells were resuspended in 400 ul of diethyl pyrocarbonate-treated water, 800 ul of RNA protect reagent (QIAGEN, Inc., Chatsworth, CA) was added, and the samples were incubated at room temperature for 5 min with vortexing for 10 s at 1-min intervals. Cells were then pelleted, resuspended in 500 ul Tris-EDTA (50:10) buffer, and transferred to 1.5-ml screw-cap tubes containing sterile glass beads (avgerage diameter, 0.1 mm; Biospec, Bartlesville, OK), 100 ul of 1% sodium dodecyl sulfate, and 650 ul of acid phenol:chloroform (5:1). The mixture was homogenized twice in a bead beater for 40 s and chilled on ice for 2 min between cycles. Samples were centrifuged for 30 min at maximum speed at 4°C. Two additional hot acid-phenol:chloroform extractions were performed by incubating the samples at 65°C for 10 min and transferring them to an ice bath for another 10 min, followed by centrifugation at maximum speed for 10 min at 4°C. The aqueous phase was collected and extracted once with chloroform:isoamyl-alcohol (24:1). RNA was precipitated with 1/10 volume of 3 M sodium acetate, pH 5, and an equal volume of isopropanol at -20°C for 1 h, followed by multiple washes with 70% ethanol, one wash with 99% ethanol, and drying in vacuo. RNA was resuspended in 52 ul of diethyl pyrocarbonate-treated water; 1 ul was used to estimate RNA concentration, and 1 ul was used to verify RNA quality in a formaldehyde gel. The remaining 50-ul sample was digested with DNase I (Ambion, Austin, TX), and then the RNA was repurified and treated on column with DNase I using an RNeasy mini kit (QIAGEN, Inc., Chatsworth, CA) as recommendedby the supplier.
Label
Cy3
Label protocol
All RNAs were purified as described above and used to generate cDNA according to the protocol provided by TIGR with the following minor modifications. The amount of RNA in each reaction was increased to 10 ug, and the molar ratio of dTTP:aa-dUTP was increased to 1:1.5. In addition, SuperscriptIII reverse transcriptase (Invitrogen, Gaithersburg, MD) was used to increase cDNA yields.
Briefly, 50-ml cultures that were intended for use in microarray experiments were grown under the desired conditions and harvested by centrifugation at 4°C. Pelleted cells were resuspended in 400 ul of diethyl pyrocarbonate-treated water, 800 ul of RNA protect reagent (QIAGEN, Inc., Chatsworth, CA) was added, and the samples were incubated at room temperature for 5 min with vortexing for 10 s at 1-min intervals. Cells were then pelleted, resuspended in 500 ul Tris-EDTA (50:10) buffer, and transferred to 1.5-ml screw-cap tubes containing sterile glass beads (avgerage diameter, 0.1 mm; Biospec, Bartlesville, OK), 100 ul of 1% sodium dodecyl sulfate, and 650 ul of acid phenol:chloroform (5:1). The mixture was homogenized twice in a bead beater for 40 s and chilled on ice for 2 min between cycles. Samples were centrifuged for 30 min at maximum speed at 4°C. Two additional hot acid-phenol:chloroform extractions were performed by incubating the samples at 65°C for 10 min and transferring them to an ice bath for another 10 min, followed by centrifugation at maximum speed for 10 min at 4°C. The aqueous phase was collected and extracted once with chloroform:isoamyl-alcohol (24:1). RNA was precipitated with 1/10 volume of 3 M sodium acetate, pH 5, and an equal volume of isopropanol at -20°C for 1 h, followed by multiple washes with 70% ethanol, one wash with 99% ethanol, and drying in vacuo. RNA was resuspended in 52 ul of diethyl pyrocarbonate-treated water; 1 ul was used to estimate RNA concentration, and 1 ul was used to verify RNA quality in a formaldehyde gel. The remaining 50-ul sample was digested with DNase I (Ambion, Austin, TX), and then the RNA was repurified and treated on column with DNase I using an RNeasy mini kit (QIAGEN, Inc., Chatsworth, CA) as recommendedby the supplier.
Label
Cy5
Label protocol
All RNAs were purified as described above and used to generate cDNA according to the protocol provided by TIGR with the following minor modifications. The amount of RNA in each reaction was increased to 10 ug, and the molar ratio of dTTP:aa-dUTP was increased to 1:1.5. In addition, SuperscriptIII reverse transcriptase (Invitrogen, Gaithersburg, MD) was used to increase cDNA yields.
Hybridization protocol
Hybridizations were carried out in the dark for 17 h at 42°C. The slides were then washed according to TIGR protocols.
Scan protocol
The slides were scanned using a GenePix Scanner (Axon Instruments Inc., Union City, CA).
Description
S. mutans manL wildtype
Data processing
After the slides were scanned, singlechannel images were loaded into TIGR Spotfinder software and overlaid. A spot grid was created according to TIGR specifications and then manually adjusted to fit all spots within the grid. The intensity values of each spot were measured and saved into “.mev” and “.tav” files. Data were normalized using LOWESS and iterative log mean centering with default settings, followed by in-slide replicate analysis using TIGR microarray data analysis software. Spots that were flagged as bad because of either low intensity values or signal saturation were automatically discarded.
