|
| Status |
Public on Nov 19, 2008 |
| Title |
HemeExcess_PgC09 |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
Total RNA, heme-excess
|
| Organism |
Porphyromonas gingivalis |
| Characteristics |
Porphyromonas gingivalis W50, grown in continuous culture under conditions of heme-excess (BHI with 5.0 µg/mL hemin). Biological replicate 2 (Each heme-excess sample is from a separate chemostat).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Samples stabilized with 0.2 Volumes of 5 % Phenol in Absolute ethanol, then pelleted by centrifugation and frozen in liquid nitrogen.
Total RNA extracted using Trizol (Invitorgen) according to manufacturers instructions but enhanced with mechanical lysis (Precellys 24 homogenizer - Bertin Technologies, Lysing Matrix B Glass Beads - MP Biomedicals). RNA was further purified using Illustra RNAspin Mini RNA Isolation kit (GE) according to manufacturers instructions including on-column DNase treatment.
|
| Label |
Cy3
|
| Label protocol |
cDNA synthesized from 10 µg total RNA using the SuperScript plus Indirect cDNA labelling system (Invitrogen) primed with 5 µg random hexamers. cDNA labelled using the Cy3 post-labelling reactive dye pack (GE) and purified using the purification module of the Invitrogen labelling system.
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| |
|
| Channel 2 |
| Source name |
Genomic DNA Reference
|
| Organism |
Porphyromonas gingivalis |
| Characteristics |
Genomic DNA from Porphyromonas gingivalis W50 used as reference sample
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA extracted using the DNeasy Blood and Tissue Kit (Qiagen) in accordance with Manufacturers instructions.
|
| Label |
Cy5
|
| Label protocol |
400 ng of genomic DNA 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.
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| |
|
| |
| Hybridization protocol |
Slides 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) denatured at 95ºC for 5 min 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 immersing 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 100 %. PMT setting adjusted to obtain a 1:1 ratio of Cy3:Cy5. Pictures of both channels saved as 16-bit tiff files
|
| Description |
No additional information
|
| 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. Spots were filtered and flagged as "bad" if circularity <60, diameter <= 50 or F pixels (area) <30. The LIMMA software package (Smyth, 2005) was used to normalize the within-array data by subtracting the "morph" background and using the "control" method to fit a global loess curve through the whole library titration series spots (MSP controls) and then applying that curve to all the other spots. Between-array normalization was also carried out for all arrays in the series using the "scale" method. Spots classified as "bad" were down-weighted to 1 % for these analyses. The "VALUE" data are the mean (as spots were printed in triplicate) of the normalized log2 (gDNA reference/ test sample) ratios.
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| |
|
| Submission date |
Oct 27, 2008 |
| Last update date |
Nov 19, 2008 |
| Contact name |
Helen Mitchell |
| Organization name |
The University of Melbourne
|
| Department |
School of Dental Science
|
| Street address |
720 Swanston St
|
| City |
Parkville |
| ZIP/Postal code |
3010 |
| Country |
Australia |
| |
|
| Platform ID |
GPL7531 |
| Series (1) |
| GSE13375 |
Porphyromonas gingivalis W50 Heme-excess vs Heme-limitation |
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