|
| Status |
Public on Jul 05, 2011 |
| Title |
Array 2-2 [ARRAY2] |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
wild type
|
| Organism |
Streptococcus pyogenes M49 591 |
| Characteristics |
serotype: M49
strain: 591
genotype/variation: wild type
|
| Growth protocol |
GAS serotype M49 and its isogenic Δralp3 mutant were grown in Todd-Hewitt broth supplemented with 0.5% yeast extract to the transition phase (OD 600nm = 1.0) at 37°C under a 5% CO2 20% O2 atmosphere.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated with FastRNA Pro Blue Kit (MP Biomedicals).
|
| Label |
Cy5
|
| Label protocol |
cDNA synthesis was performed with 10 µg of total RNA. The RNA was mixed with ramdom primers (Promega) and spike-ins (Two-Color RNA Spike-In Kit, Agilent Technologies) followed by the incubation at 70°C for 10 min and 5 min on ice. Then, the following reagents were added: 10 µl of 5x First Strand Buffer (Invitrogen), 5 µl of 0.1 M DTT (Invitrogen), 0.5 µl of a dNTP mix (10 mM dATP, dGTP, and dTTP, 2.5 mM dCTP), 1.25 µl of Cy3-dCTP or Cy5-dCTP (GE Healthcare), and 2 µl of the SuperScript II reverse transcriptase (Invitrogen). The mixture was incubated at 42°C for 60 min and then heated to 70°C for 10 min, followed by incubation on ice for 5 min. Then, the RNA was degraded by incubation with 2 units of RNaseH (Invitrogen) at room temperature for 30 min and labeled cDNA was purificated using the CyScribe GFX Purification Kit (GE Healthcare).
|
| |
|
| Channel 2 |
| Source name |
Δralp3
|
| Organism |
Streptococcus pyogenes M49 591 |
| Characteristics |
serotype: M49
strain: 591
genotype/variation: Δralp3
|
| Growth protocol |
GAS serotype M49 and its isogenic Δralp3 mutant were grown in Todd-Hewitt broth supplemented with 0.5% yeast extract to the transition phase (OD 600nm = 1.0) at 37°C under a 5% CO2 20% O2 atmosphere.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated with FastRNA Pro Blue Kit (MP Biomedicals).
|
| Label |
Cy3
|
| Label protocol |
cDNA synthesis was performed with 10 µg of total RNA. The RNA was mixed with ramdom primers (Promega) and spike-ins (Two-Color RNA Spike-In Kit, Agilent Technologies) followed by the incubation at 70°C for 10 min and 5 min on ice. Then, the following reagents were added: 10 µl of 5x First Strand Buffer (Invitrogen), 5 µl of 0.1 M DTT (Invitrogen), 0.5 µl of a dNTP mix (10 mM dATP, dGTP, and dTTP, 2.5 mM dCTP), 1.25 µl of Cy3-dCTP or Cy5-dCTP (GE Healthcare), and 2 µl of the SuperScript II reverse transcriptase (Invitrogen). The mixture was incubated at 42°C for 60 min and then heated to 70°C for 10 min, followed by incubation on ice for 5 min. Then, the RNA was degraded by incubation with 2 units of RNaseH (Invitrogen) at room temperature for 30 min and labeled cDNA was purificated using the CyScribe GFX Purification Kit (GE Healthcare).
|
| |
|
| |
| Hybridization protocol |
200 ng of Cy3-labeled cDNA and 200 ng of Cy5-labeled cDNA were hybridizied together to the microarray (Custom Array GE 8x15K; Agilent). After hybridization, slides were washed sequential.
|
| Scan protocol |
Scanned on an Agilent G2565AA scanner. Images were quantified using Agilent Feature Extraction Software (version 10.7.3.1).
|
| Description |
Array 2
Biological raplicate 2 of 4
mic_252555310005_S03_Green_Cropped_GE2_107_Sep09_2_2.txt
|
| Data processing |
data was analyzed using Gene Spring GX 11.0.2 software.
|
| |
|
| Submission date |
Jul 05, 2011 |
| Last update date |
Jul 05, 2011 |
| Contact name |
Nikolai Siemens |
| E-mail(s) |
[email protected]
|
| Phone |
00493814945939
|
| Organization name |
University of Rostock
|
| Department |
IMIKRO
|
| Lab |
Kreikemeyer
|
| Street address |
Schillingallee 70
|
| City |
Rostock |
| State/province |
MV |
| ZIP/Postal code |
18057 |
| Country |
Germany |
| |
|
| Platform ID |
GPL13816 |
| Series (1) |
| GSE30397 |
GAS M49: wild type vs Δralp3 mutant |
|
