|
| Status |
Public on Mar 20, 2017 |
| Title |
Planktonic 3 |
| Sample type |
RNA |
| |
|
| Source name |
Porphyromonas gingivalis culture 96h
|
| Organism |
Porphyromonas gingivalis |
| Characteristics |
sampel type: planktonic
|
| Treatment protocol |
Plates were incubated in anaerobic conditions at 37°C for 96 h
|
| Growth protocol |
Planktonic cultures of P. gingivalis were grown anaerobically at 37ºC for 24 h in a protein-rich medium containing brain-heart infusion (BHI)
A volume of 1.5 mL of P. gingivalis inoculums (concentration 10^8 CFU/mL) was placed in pre-sterilized polystyrene 24-well tissue culture plates (Greiner Bio-one) with or without the presence of sterile ceramic calcium hydroxyapatite discs
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After 96 h of incubation, P. gingivalis planktonic and biofilm 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 3 sample 3 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 |
Mar 17, 2017 |
| Last update date |
Mar 21, 2017 |
| Contact name |
Patricia Romero Lastra |
| E-mail(s) |
patrrome@ucm.es
|
| 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 |
GPL23193 |
| Series (1) |
| GSE96756 |
Comparative gene expression analysis of Porphyromonas gingivalis ATCC 33277 in planktonic and biofilm states |
|
