|
| Status |
Public on Jun 05, 2019 |
| Title |
PgP_A1: PlanktonicPg_A1 |
| Sample type |
RNA |
| |
|
| Source name |
Planktonic culture of P. gingivalis ATCC 33277
|
| Organism |
Porphyromonas gingivalis ATCC 33277 |
| Characteristics |
strain: ATCC 33277
tissue: Planktonic culture of P. gingivalis ATCC 33277
|
| Treatment protocol |
Using pre-sterilized polystyrene 24-well tissue culture plates (Greiner Bio-one, Frikenhausen, Germany), two types of growing conditions were developed: (1) The test group (P. gingivalis growing within a multispecies biofilm). In each well of the plate, 1.5 mL of the mix solution containing the six reference strains, were deposited together with a sterile ceramic calcium hydroxyapatite discs (HA) [7-mm diameter (standard deviation, SD = 0.2 mm) and 1.8 mm thickness (Clarkson Chromatography Products, Williamsport, PA, USA)]; (2) The control group (P. gingivalis growing planktonically). In each well, a volume of 1.5 mL of pure culture of P. gingivalis (10^8 CFUs/mL) was deposited in the absence of discs.
Plates were incubated in anaerobic conditions at 37°C for 96 h
|
| Growth protocol |
Planktonic cultures of P. gingivalis ATCC 33277, Streptococcus oralis CECT 907T, Actinomyces naeslundii ATCC 19039, Veillonella parvula NCTC 11810, Fusobacterium nucleatum DMSZ 20482 and Aggregatibacter actinomycetemcomitans DSMZ 8324 were grown anaerobically at 37ºC for 24 h in a protein-rich medium containing brain-heart infusion (BHI)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After 96 h of incubation, both types of samples (multispecies biofilm and planktonic P. gingivalis) cells were harvested
Total RNA was extracted from the harvested samples listed above, using the TRIzol® Max Bacterial RNA Isolation Kit (Ambion, Life Technologies, Carlsbad, CA, USA)
RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Fluorescently labeled cDNA for microarray hybridizations was obtained by using the SuperScript Indirect cDNA Labeling System (Invitrogen)
|
| |
|
| Hybridization protocol |
Preparation of probes and hybridization was performed as described (One-Color Microarray Based Gene Expression Analysis Manual Ver. 6.5, Agilent Technologies)
For each hybridization, 600 ng of Cy3 probes were mixed and added to 5 µL of 10x Blocking Agent and Nuclease free water in a 25 µL reaction
Then, 25 µL from 2x GExHybridization buffer was added and mixed carefully
The samples were placed on ice and quickly loaded onto arrays, hybridized at 65ºC for 17 h in a rotating Agilent hybridization oven
After hybridization, microarrays were washed once in GE wash buffer 1 at room temperature (1 min) and once in GE Wash Buffer 2 at 37ºC (1 min).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner using one color scan setting for 8x15K array slides
|
| Description |
Gene expression after 96h Planktonic 1 sample 1 of 3
|
| Data processing |
Images from Cy3 channel were equilibrated and captured with a high-resolution scanner (Agilent) and spots quantified using Feature Extraction software (Agilent)
Background correction and normalization of data expression were performed using LIMMA
|
| |
|
| Submission date |
Jun 04, 2019 |
| Last update date |
Jun 05, 2019 |
| Contact name |
Patricia Romero Lastra |
| E-mail(s) |
[email protected]
|
| Organization name |
Universidad Complutense de Madrid
|
| Street address |
Plaza de Ramon y Cajal s/n
|
| City |
Madrid |
| ZIP/Postal code |
28040 |
| Country |
Spain |
| |
|
| Platform ID |
GPL26732 |
| Series (1) |
| GSE132157 |
Gene expression of Porphyromonas gingivalis ATCC 33277 when growing in an in vitro multispecies biofilm |
|
