strain: atcc 25586 wild type aggregation status: aggregated
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 1:100 in 20 mL of SDM and grown 16 hrs overnight to late log phase (OD600 0.7 – 0.9). The following day, the cells were centrifuged and resuspended to OD600 0.5 – 0.6 in either 25 mL SDM, SDM + 25% saliva, or SDM + 25% saliva and 50 mM L-lysine. Next, the cells were prewarmed to 37°C before incubating anaerobically for 90 min. at 37°C. Every 15 – 20 min., the non-aggregating cultures (SDM and SDM + saliva/lysine) were gently agitated to maximize separation between the cells and prevent any settling.
Extracted molecule
total RNA
Extraction protocol
After the assay period, the cells were immediately centrifuged at 4°C in a prechilled refrigerated centrifuge. Cell pellets were resuspended in 500 µL chilled TE buffer on ice. Next, the cells were placed in a 70°C waterbath and 900 µL preheated acidic phenol (pH 4.3) was immediately added to the cultures followed by SDS to a final concentration of 1% wt/vol. The mixture was incubated for 5 minutes before removing, adding 400 µL chloroform, and centrifuging. The mixture was further phenol/chloroform extracted 3 times before the RNA was precipitated and resuspended in 90 µL RNase-free water + 10 µL DNase buffer. DNase was added to the samples and incubated at 37°C for 45 minutes. Finally, the samples were further purified with Qiagen RNeasy MinElute spin columns according to the manufacturer’s protocol and eluted in RNase-free water. The resulting RNA integrity was confirmed by the presence of clearly defined rRNA bands in agarose gels and later quantified for concentration by OD260 readings.
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 the GeneChip® operating system 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.
