For gene-array analysis, a 4 ml aliquot from a 3 days liquid culture (per 1 L: 15 g trypticase peptone, 5 g neutralized soja peptone, 5 g sodium chloride, 5 g yeast extract, 0.5 g L-cysteine, supplemented with 10 mg vitamin K and 5 mg hemin) was inoculated into fresh medium to render an OD660 0.05 and cultivated for 12 h. Using this culture, 4 ml with an OD660 value of 0.1 were incubated with 100 µg/ml Myrothamnus flabellifolia or medium alone (control group) for 6 h.
Growth protocol
P. gingivalis (ATCC 33277) was cultured under anaerobic conditions (Anaerocult, Merck, Darmstadt, Germany) at 37°C in medium comprised of 15 g trypticase peptone, 5 g neutralized soja peptone, 5 g sodium chloride, 5 g yeast extract, 0.5 g L-cysteine per litre, and supplemented with 10 mg vitamin K and 5 mg hemin.
Extracted molecule
total RNA
Extraction protocol
Harvested cells were resuspended in PBS (100 µl) and mixed with TE-Buffer (500 µl), 1% SDS, phenol and lysing matrix B (0,4 g) (MP Biomedicals, Irvine, USA). Cells were lysed by FastPrep®-24-System (MP Biomedicals, Irvine, USA) (2 × 50 sec, 5.0 m/s2) and RNA-isolation was followed by phenol-chloroform-extraction and ethanol-precipitation. Removal of DNA was performed by NucleoSpin®rDNase (Machery-Nagel, Düren, Germany) according to the manufacturer's instructions.
Label
Cy3
Label protocol
Labeling with Cy3 was executed as recommended by the array user guide (Gene Expression Analysis v 2.0, NimbleGen).
Hybridization protocol
Hybridization was executed as recommended by the array user guide (Gene Expression Analysis v 2.0, NimbleGen).
Scan protocol
Scanning was performed with the DNA Microarray Scanner (Agilent) at a resolution of 5 µm and NimbleScanTM version 2.4 software.
Description
This sample is of untreated wild-type Porphyromonas gingivalis ATCC 33277. The RNA was pooled from two independent biological replicates, each from separate cultures.
Data processing
The raw data (.pair files) were checked for inhomogenous hybridization. Quantile normalization was performed as implemented by GeneSpring GX 11.0, Agilent Technologies.
Polyphenols from Myrothamnus flabellifolia Welw. inhibit in vitro adhesion of Porphyromonas gingivalis and exert anti-inflammatory cytoprotective effects in KB cells
Data table header descriptions
ID_REF
VALUE
Quantile-normalized, averaged gene-signal intensities in log2 scale
