|
| Status |
Public on May 30, 2008 |
| Title |
P. gingivalis W83 polyP75 2h |
| Sample type |
RNA |
| |
|
| Source name |
total RNA from Porphyromonas gingivalis W83
|
| Organism |
Porphyromonas gingivalis |
| Characteristics |
strain W83
|
| Biomaterial provider |
State University of New York at Buffalo
|
| Treatment protocol |
Culture of Porphyromonas gingivalis W83 grown to early-exponential phase(O.D600=0.3), was divided in half. One aliquot was left untreated while the other was treated with 0.03% polyphosphate with a chain length of 75 (polyP 75; Sigma). Both the bacterial aliquots were incubated for 2h under anaerobic conditions (5% CO2, 10% H2, 85% N2).
|
| Growth protocol |
P. gingivalis strain W83 was grown on blood agar plates consisting of 3% Tryptic soy agar, 0.5% yeast extract, 5% sheep blood, 5 μg/ml of hemin, and 0.4 μg/ml of vitamin K1. P. gingivalis was also cultured in half-strength brain heart infusion broth supplemented with 0.5% yeast extract, 5 μg/ml of hemin, and 1 μg/ml of vitamin K1. Cells were grown and maintained at 37℃ under anaerobic conditions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
10ml of each culture was mixed with 5% water-saturated phenol (pH 4.3) in ethanol. The cells were harvested by centrifugation and total RNA was extracted using Trizol (Invitrogen) following manufacture's instructions. DNA was removed by treatment with RNase-free DNase I.
|
| Label |
Cy3
|
| Label protocol |
20μg of each total RNA was converted into cDNA using the Superscript II cDNA Conversion Kit (Invitrogen). Double-stranded cDNA was random-prime labeled with Cy3-nonamers.
|
| |
|
| Hybridization protocol |
Two individual Cy3-labeled cDNA samples originating from W83 treated or untreated with polyP75 were hybridized to the NimbleChip gene expression arrays(A4387-00-01). Hybridizations were carried out for 16h at 42℃
|
| Scan protocol |
The arrays were analyzed using an Axon GenePix 4000B microarray scanner with associated software (Molecular Devices Corp.). Gene expression levels were calculated with NimbleScan Version 2.3 (NimbleGen). Relative signal intensities for each gene were generated using the Robust Multi-Array Average algorithm.
|
| Description |
The whole genome of 1,909 genes of P. gingivalis W83 (GenBank accession no. NC_002950) was submitted to NimbleGen System Inc. for microarray design and manufacture using maskless, digital micromirror technology. Five replicates of the genome were included per chip. An average of 19 different 60-base oligonucleotides (60-mer probes) represented each gene in the genome. Sixty-mer probes were selected such that each probe had at least three mismatches compared to all other 60-mers in the target genome.
|
| Data processing |
The data were processed based on quantile normalization method using the R package. This normalization method aims to make the distribution of intensities for each array in a set of arrays the same. The background-adjusted, normalized, and log transformed intensity values were then analyzed using GeneSpring GX 7.3.1 (Silicon Genetics).
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| |
|
| Submission date |
May 16, 2008 |
| Last update date |
May 29, 2008 |
| Contact name |
Ji-Hoi Moon |
| E-mail(s) |
prudence75@khu.ac.kr
|
| Phone |
+82-2-961-0598
|
| Fax |
+82-2-960-2838
|
| Organization name |
School of Dentistry, Kyung Hee University
|
| Department |
Oral Microbiology
|
| Street address |
1 Hoeki-Dong, Dongdaemoon-Ku
|
| City |
Seoul |
| ZIP/Postal code |
130-701 |
| Country |
South Korea |
| |
|
| Platform ID |
GPL6533 |
| Series (1) |
| GSE11471 |
Polyphosphate affects gene expression and viability of Porphyromonas gingivalis W83 |
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