|
| Status |
Public on Sep 15, 2009 |
| Title |
MS11 vs. hfq (III) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
MS11
|
| Organism |
Neisseria gonorrhoeae |
| Characteristics |
genotype: wildtype (MS11)
biological replicate: III
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Qiagen RNeasy including on-column DNase-digest
|
| Label |
Cy3
|
| Label protocol |
Equal amounts (10 µg) of total bacterial RNA were reverse transcribed. cDNA was labeled with Cy3 or Cy5 using the CyScribe Postlabelling Kit (GE Healthcare) and labeled cDNA purified using the CyScribe GFX purification kit (GE Healthcare)
|
| |
|
| Channel 2 |
| Source name |
hfq
|
| Organism |
Neisseria gonorrhoeae |
| Characteristics |
genotype: hfq KO mutant (MS11hfq)
biological replicate: III
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Qiagen RNeasy including on-column DNase-digest
|
| Label |
Cy5
|
| Label protocol |
Equal amounts (10 µg) of total bacterial RNA were reverse transcribed. cDNA was labeled with Cy3 or Cy5 using the CyScribe Postlabelling Kit (GE Healthcare) and labeled cDNA purified using the CyScribe GFX purification kit (GE Healthcare)
|
| |
|
| |
| Hybridization protocol |
Labeled samples was mixed and hybridized to 8x15K custom-made microarray according to the supplier`s protocol (Agilent Technologies). Washing of the hybridized arrays was done with the SSC protocol according supplier`s protocol (Agilent Technologies).
|
| Scan protocol |
Scanning of microarrays was performed with 5 µm resolution using a DNA microarray laser scanner (Agilent Technologies).
|
| Description |
Features were extracted with an image analysis tool version A.9.5.3 using the GE2-V5_95_Feb07 protocol (Agilent Technologies).
|
| Data processing |
Data analysis was carried out on the Rosetta Inpharmics platform Resolver Version 7.0. Ratio profiles were combined in an error-weighted fashion with Resolver to create ratio experiments. A 1.75-fold change expression cut-off for ratio experiments was applied together with anti-correlation of ratio profiles rendering the microarray analysis set highly significant (P-value > 0.05), robust and reproducible. The LogRatio (base 10) represents Cy5/Cy3 for each sample pair. Values were generated with FE 9.5.3.1. software (Agilent Technologies) using the GE2-v4_95_Feb07 protocol and default settings. In brief, background, signal biases and errors were calculated applying Signal Correction - Calculate Surface Fiting, Spatial Detrending and the Most Conservative error model. Dye biases were corrected with the Dye Normalization Probe Selection Method using Rank Consistent Probes and the normalization correction methods Linear and Lowess. To determine if a feature is differentially expressed, three measures are important: LogRatio, LogRatioError and the PValueLogRatio. The LogRatio (VALUE) is the log (base 10) of the ratio of the red channel signal to the green channel signal after background adjustment and dye bias correction. LogRatioError is the error about the mean of the log ratio. PValueLogRatio is a measure of confidence that a feature was identified as being differentially expressed. The log ratio error was determined by the error model and therefore was used to help assess the significance of the computed log ratio. The p-value was calculated using the logratio and the log ratio error. Genes with lower p-values have higher confidence of being differentially expressed. In order to combine dye-reversal color-swaps, a negative polarity was added to one of the arrays.
|
| |
|
| Submission date |
May 11, 2009 |
| Last update date |
Sep 15, 2009 |
| Contact name |
Hans-Joachim Mollenkopf |
| E-mail(s) |
mollenkopf@mpiib-berlin.mpg.de
|
| Phone |
+49 30 28460 482
|
| Organization name |
Max-Planck-Institute for Infection Biology
|
| Lab |
Microarray/Genomics Core Facility
|
| Street address |
Charitéplatz 1
|
| City |
Berlin |
| ZIP/Postal code |
10117 |
| Country |
Germany |
| |
|
| Platform ID |
GPL7101 |
| Series (1) |
| GSE16046 |
The effect of hfq on global gene expression and virulence in Neisseria gonorrhoeae |
|
