|
| Status |
Public on Dec 31, 2021 |
| Title |
feoB_mutant_rep1 |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
FeoB mutant
|
| Organism |
Porphyromonas gingivalis |
| Characteristics |
strain/background: W50
genotype/variation: FeoB mutant
chemostat: D
biological replicate: 1
|
| Growth protocol |
Porphyromonas gingivalis FeoB mutant of W50 grown in continuous culture under conditions of heme-excess (BHI with 5 µg/mL hemin).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Samples were stabilized with 0.2 volumes of 5% phenol in absolute ethanol, then pelleted by centrifugation and frozen in liquid nitrogen. Total RNA was extracted using Trizol (Invitrogen) according to the manufacturer's instructions but enhanced with mechanical lysis (Precellys 24 homogenizer - Bertin Technologies, Lysing Matrix B Glass Beads - MP Biomedicals). RNA was further purified using the Illustra RNAspin Mini RNA Isolation kit (GE) according to the manufacturer's instructions including on-column DNase treatment.
|
| Label |
Cy3
|
| Label protocol |
cDNA was synthesized from 10 µg total RNA using the SuperScript Plus Indirect cDNA labelling system (Invitrogen) primed with 5 µg random hexamers. cDNA was labelled using the Cy3 post-labelling reactive dye pack (GE) and purified using the purification module of the Invitrogen labelling system.
|
| |
|
| Channel 2 |
| Source name |
Genomic DNA reference
|
| Organism |
Porphyromonas gingivalis W50 |
| Characteristics |
strain/background: W50
|
| Growth protocol |
Porphyromonas gingivalis W50 wild type grown in continuous culture under conditions of heme-excess (BHI with 5 µg/mL hemin).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen) in accordance with the manufacturer's instructions.
|
| Label |
Cy5
|
| Label protocol |
400 ng of genomic DNA was used to synthesize dUTP-incorporated DNA with the BioPrime Plus Array CGH Indirect Genomic Labelling System (Invitrogen), and then labelled using the Cy5 post-labelling reactive dye pack (GE) and purified using the purification module of the Invitrogen labelling system.
|
| |
|
| |
| Hybridization protocol |
Slides were hybridised using either heme-excess or heme-limited samples labelled with Cy3, combined with a universal genomic reference labelled with Cy5. Prior to hybridisation, microarray slides were immersed for 1 h in blocking solution (35% formamide, 1% BSA, 0.1% SDS, 5X SSPE [1X SSPE is 150 mM NaCl, 10 mM NaH2PO4, 1 mM EDTA]) at 42ºC. After blocking, slides were briefly washed in H2O followed by 99% ethanol and then dried by centrifugation. Labelled cDNAs were resuspended in 55 µL of hybridization buffer (35% formamide, 5X SSPE, 0.1% SDS, 0.1 mg/mL Salmon Sperm DNA) and denatured at 95ºC for 5 min, and then applied to slides and covered with LifterSlips (Erie Scientific). Hybridisation was performed at 42ºC for 16 h. Following hybridisation, slides were successively washed in 0.1% SDS plus 2X SSC [1X SSC is 150 mM NaCl 15 mM sodium citrate] (5 min at 42 ºC, all further washes performed at room temperature), 0.1% SDS plus 0.1X SSC (10 min), 0.1X SSC (4 washes, 1 min each), and then quickly immersed in 0.01X SSC, then 99% ethanol and using centrifugation to dry the slides.
|
| Scan protocol |
GenePix 4000B microarray scanner (Molecular Devices) at 532 nm (Cy3) and 635 nm (Cy5) with a 10 µm resolution and laser power at 10 %. PMT setting adjusted to obtain a 1:1 ratio of Cy3:Cy5. Pictures of both channels saved as 16-bit tiff files.
|
| Description |
FB1_PgA18
|
| Data processing |
Image analysis was performed using the GenePix Pro 6.0 software (Molecular Devices), and “morph” background values were used as the background estimates in further analysis. The LIMMA software package (Smyth, 2005) was used to normalize the within-array data by subtracting the morph background and using the loess method to fit a global loess curve. Between-array normalization was also carried out for all arrays in the series using the VSN method.
|
| |
|
| Submission date |
Apr 05, 2012 |
| Last update date |
Dec 31, 2021 |
| Contact name |
Catherine Butler |
| E-mail(s) |
cbutler@unimelb.edu.au
|
| Phone |
61-3-93411565
|
| Organization name |
The University of Melbourne
|
| Department |
The Melbourne Dental School
|
| Lab |
The Reynolds Lab
|
| Street address |
720 Swanston St
|
| City |
Carlton |
| State/province |
VIC |
| ZIP/Postal code |
3053 |
| Country |
Australia |
| |
|
| Platform ID |
GPL15412 |
| Series (1) |
| GSE37072 |
Porphyromonas gingivalis W50 wild type vs. FeoB mutant |
|
