Cells were harvested by centrifugation and lysed using a beat-beater.
Growth protocol
Cells were first grown to stationary phase in Columbia broth, which was then used as a starter culture to inoculate SDM cultures. Stationary phase cells were diluted 1:200 in 20 mL of SDM and grown 16 hrs overnight to early log phase (OD600 0.3 – 0.5). The following day, the 9% and 33% sheep blood was added to some cultures before incubating anaerobically for 3h at 37°C.
Extracted molecule
total RNA
Extraction protocol
Briefly, the cell pellets were resuspended in chilled Tris-EDTA buffer on ice and transferred to a 2-ml screw-cap tube containing 500 μl 0.1-mm silica beads. 900μl of preheated acidic phenol (pH 4.3) and 140μl of 10% sodium dodecyl sulfate were immediately added to the mixture and submitted to two consecutive 30-s homogenization cycles with a FastPrep-24 system set at a speed of 6.0 M/s, followed by acidic phenol-chloroform extraction and isopropyl alcohol precipitation. After treatment with RNase-free DNase, RNA was further purified over RNeasy spin columns. The resulting RNA integrity was confirmed by the presence of clearly defined rRNA bands in agarose gels.
Label
Biotin
Label protocol
32 µL of fragmented cDNA was labeled with the BioArray terminal labeling kit (Enzo, New York, NY) in a total volume of 50 µL containing 10 µL 5x buffer, 5 µL 10x CoCl2, and 2 µL terminal deoxynucleotide transferase. The reaction was incubated at 37°C for 60 min.
Hybridization protocol
The hybridization and washing procedures were performed similarly as recommended in the GeneChip® Expression Analysis Technical Manual with some minor modifications. The hybridization buffer consisted of 1x MES buffer (100 mM morpholineethanesulfonic acid [MES], 1M NaCl, 20 mM EDTA, 0.01% Tween-20), 50 pM B2 control oligo, 0.1 mg/mL herring sperm DNA, 0.5 mg/mL BSA, and the fragmented, labeled cDNA in a total volume of 200 µL. The hybridization solution was then loaded into the microarray cartridge and mixed for 16 hr at 60 rpm and 47°C in an Affymetrix hybridization oven. After the incubation period, the hybridization solution was removed and the array was washed and stained in the Affymetrix Fluidics Station 450 using a slightly modified script of the standard wash script file “ProkGE-WS2v3-450”, which increased the wash stringency from 45°C to 47°C. The staining solution consisted of 3 different MES buffers each in a 600 µL total volume. The first consisted of 1x MES buffer, 2 mg/mL BSA, and 10 µg/mL streptavidin, the second consisted of 1x MES buffer, 2 mg/mL BSA, 0.1 mg/mL goat IgG, and 5 µg/mL biotin anti-streptavidin, and the third solution consisted of 1x MES buffer, 2 mg/mL BSA, and 10 µg/mL streptavidin-phycoerythrin (Molecular Probes, Eugene, OR).
Scan protocol
The GeneChips® were scanned at 570 nM using an Affymetix 7G laser scanner.
Description
Standard 49 format custom GeneChip.
Data processing
Analysis of signal intensities was performed using GeneChip® Operating Software (GCOS) ver. 1.4, and gene expression data were compared using the GCOS batch analysis function. Normalization procedures are performed directly by the software using a script designed by Affymetrix and provided with the F. nucleatum custom array. This eliminates user bias during the normalization procedure.
