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Status |
Public on Aug 21, 2013 |
Title |
Slide 27_Aerobic culture_iCORM-3_biol rep 2_Cy3 5 Cy5 0 |
Sample type |
RNA |
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Channel 1 |
Source name |
Continuous aerobically grown cultures in Evans medium, prior to CORM-3 addition
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Organism |
Escherichia coli |
Characteristics |
strain: Wild type strain MG1655
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
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Label |
Cy5
|
Label protocol |
cDNA synthesis was carried out using 16 μg of RNA, primed with 5 μg Random Primers (Invitrogen). Reaction mixes (30 μl) containing 0.5 mM dATP, dTTP, and dGTP, 0.2 mM dCTP, and 67 μM Cy-3 or Cy-5-dCTP were incubated at 25 °C for 5 min, then at 50 °C overnight with 300 U of Superscript III Reverse Transcriptase (Invitrogen). Following synthesis, cDNA was purified using a PCR purification kit (Qiagen).
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Channel 2 |
Source name |
Continuous aerobically grown cultures in Evans medium, exposed to 40uM iCORM-3 for 5min
|
Organism |
Escherichia coli |
Characteristics |
strain: Wild type strain MG1655
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
Label |
Cy3
|
Label protocol |
cDNA synthesis was carried out using 16 μg of RNA, primed with 5 μg Random Primers (Invitrogen). Reaction mixes (30 μl) containing 0.5 mM dATP, dTTP, and dGTP, 0.2 mM dCTP, and 67 μM Cy-3 or Cy-5-dCTP were incubated at 25 °C for 5 min, then at 50 °C overnight with 300 U of Superscript III Reverse Transcriptase (Invitrogen). Following synthesis, cDNA was purified using a PCR purification kit (Qiagen).
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Hybridization protocol |
For hybridization to the microarray slides, 400 ng cDNA was added to 25 μl 2 x Hi-RMP Hybridization Buffer and 5 μl 10 x Gene Expression Blocking Agent (Agilent Technologies) to give 50 μl total volume. Custom Gene Expression Microarray slides each containing 8 x 15K arrays were purchased from Agilent Technologies. For hybridization one 8 x Gasket Slide was placed inside a Microarray Hybridization Chamber and samples loaded for 8 different conditions, followed by placement of the microarray slide over the gasket. After sealing the chamber the arrays were incubated at 65 °C, rotating at 10 rpm for ~17 h. Following incubation, the slides were washed in Gene Expression Wash Buffer 1 for 1 min, followed by washing in Gene Expression Wash Buffer 2 for 1 min. Slides were dried and scanned in a microarray scanner (Agilent).
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Scan protocol |
Scanned on an Agilent DNA microarray scanner with SureScan high-resolution technology
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Description |
The labelling procedure uses equal quantities of RNA from adequate CORM-3 samples and samples prior to addition of CORM-3
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Data processing |
Agilent Feature Extraction Software (v 10.10.1.1) was used for background subtraction and LOWESS normalization.
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Submission date |
Sep 12, 2012 |
Last update date |
Aug 21, 2013 |
Contact name |
Robert Poole |
E-mail(s) |
R.poole@shef.ac.uk
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Phone |
01142224447
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Organization name |
University of Sheffield
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Department |
Molecular Biology & Biotechnology
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Lab |
F13
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Street address |
Firth Court, Western Bank
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City |
Sheffield |
State/province |
South Yorkshire |
ZIP/Postal code |
S10 2TN |
Country |
United Kingdom |
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Platform ID |
GPL16048 |
Series (1) |
GSE40811 |
Transcriptional profiling of Escherichia coli after addition of CORM-3 and iCORM-3 to aerobically and anaerobically growing cells |
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