|
| Status |
Public on Mar 29, 2010 |
| Title |
Rp22 Vs Erus replicate 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Rp22 replicate 3
|
| Organism |
Rickettsia prowazekii str. Rp22 |
| Characteristics |
Purifed bacteria by lysing the infected cells with 1% trypsin and cell debris were eliminated by successive centrifugations at 1000 rpm. Bacteria were stored at -80°C before RNA extraction
|
| Growth protocol |
Growth in L929 at 32°C with EMEM medium with 4% SVF and 1% L-glutamine
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extraction and purification from purified bacteria was carried out using the RNeasy kit (Qiagen) with some modifications. Briefly, pellet of bacteria were resuspended with 100µl of TE + Lyzoyme (10mg/ml) and incubated for 10 min at RT.Then The amount and quality of obtained RNA were determined with the microfluidic-based platform (Agilent 2100 Bioanalyzer) and using the RNA 6000 Nano Labchip kit (Agilent). cDNA was synthesized as described by La et al. (2008) using the M-MLV Reverse Transcriptase (Invitrogen, Cergy-Pontoise, France).
|
| Label |
Cy5
|
| Label protocol |
Twenty nanograms of cDNA were amplified using the GenomiPhi DNA amplication kit (Amersham Biosciences, Uppsala, Sweden) and labeled with Cy3-dCTP or Cy5-dCTP dyes (Amersham Biosciences) using the BioPrime DNA labeling System (Invitrogen, Cergy-Pontoise, France). Following purification with Qiaquick columns (Qiagen), the levels of Cy3-dCTP and Cy5-dCTP incorporation were quantified by absorbance measurement at 550 nm and 650 nm, respectively.
|
| |
|
| Channel 2 |
| Source name |
Erus replicate 3
|
| Organism |
Rickettsia prowazekii |
| Characteristics |
Purifed bacteria by lysing the infected cells with 1% trypsin and cell debris were eliminated by successive centrifugations at 1000 rpm. Bacteria were stored at -80°C before RNA extraction
|
| Treatment protocol |
Total RNA extraction and purification from purified bacteria was carried out using the RNeasy kit (Qiagen) with some modifications. Briefly, pellet of bacteria were resuspended with 100µl of TE + Lyzoyme (10mg/ml) and incubated for 10 min at RT.Then The amount and quality of obtained RNA were determined with the microfluidic-based platform (Agilent 2100 Bioanalyzer) and using the RNA 6000 Nano Labchip kit (Agilent). cDNA was synthesized as described by La et al. (2008) using the M-MLV Reverse Transcriptase (Invitrogen, Cergy-Pontoise, France).
|
| Growth protocol |
Growth in L929 at 32°C with EMEM medium with 4% SVF and 1% L-glutamine
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extraction and purification from purified bacteria was carried out using the RNeasy kit (Qiagen) with some modifications. Briefly, pellet of bacteria were resuspended with 100µl of TE + Lyzoyme (10mg/ml) and incubated for 10 min at RT.Then The amount and quality of obtained RNA were determined with the microfluidic-based platform (Agilent 2100 Bioanalyzer) and using the RNA 6000 Nano Labchip kit (Agilent). cDNA was synthesized as described by La et al. (2008) using the M-MLV Reverse Transcriptase (Invitrogen, Cergy-Pontoise, France).
|
| Label |
Cy3
|
| Label protocol |
Twenty nanograms of cDNA were amplified using the GenomiPhi DNA amplication kit (Amersham Biosciences, Uppsala, Sweden) and labeled with Cy3-dCTP or Cy5-dCTP dyes (Amersham Biosciences) using the BioPrime DNA labeling System (Invitrogen, Cergy-Pontoise, France). Following purification with Qiaquick columns (Qiagen), the levels of Cy3-dCTP and Cy5-dCTP incorporation were quantified by absorbance measurement at 550 nm and 650 nm, respectively.
|
| |
|
| |
| Hybridization protocol |
Hybridizations were performed for 17 h at 60°C in dedicated microchambers with 50 pmol of both control or eschar samples. Stringent washings were then performed according to manufacturer’s instructions.
|
| Scan protocol |
Slides were scanned with XDR range at a 5-µm resolution. Feature Extractor 7.1 (Agilent Technologies) was used for image analysis.
|
| Description |
Comparison of biological replicate 3
|
| Data processing |
Data filtering and normalisation were then performed using the Midas module of TM4. The background-subtracted signals were normalised by local substraction method, and intensity signals were normalised by global lowess method. Normalised signals were used for analysis with the TMev module of TM4.
|
| |
|
| Submission date |
Apr 10, 2009 |
| Last update date |
Mar 29, 2010 |
| Contact name |
LEROY Quentin |
| E-mail(s) |
[email protected]
|
| Organization name |
URMITE
|
| Street address |
27 Boulevard Jean Moulin
|
| City |
Marseille |
| ZIP/Postal code |
13005 |
| Country |
France |
| |
|
| Platform ID |
GPL8427 |
| Series (1) |
| GSE15630 |
Multiomics study to identify virulence factors of Rickettsia prowazekii revealed its adaptive mutation capabilities |
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