|
| Status |
Public on Aug 10, 2009 |
| Title |
ME0053 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Escherichia coli O157 TUV93-0 MEM-HEPES + 20uM ME0053 (in DMSO)
|
| Organism |
Escherichia coli |
| Characteristics |
strain: O157:H7 EHEC TUV93-0
od: 0.7
media: MEM-HEPES supplemented with 0.1% Glucose and 250nM Fe(NO3)2
inhibitor: 20uM ME0053
|
| Treatment protocol |
Escherichia coli O157 TUV93-0 was grown in the presence of 20uM ME0053
|
| Growth protocol |
Escherichia coli O157 TUV93-0 was grown at 37 degrees C to OD 0.7 in MEM-HEPES supplemented with 0.1% Glucose and 250nM Fe(NO3)2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
An RNeasy minikit was used to prepare total RNA according to the manufacturer’s instructions (QIAGEN Ltd.). Any contaminating DNA was removed using a DNAase column kit (QIAGEN Ltd).
|
| Label |
Cy5
|
| Label protocol |
20 ug total RNA was converted to Cy3-labelled cDNA
using the CyScribe Post-Labelling Kit (Amersham Biosciences/GE
Healthcare) according to manufacturer’s instructions. Amino-allyllabelled
nucleotides were incorporated into the cDNA by reverse
transcription of the total RNA, followed by a direct chemical coupling
of Cy3 NHS-dye esters to the amino-allyl-labelled
cDNAs.
|
| |
|
| Channel 2 |
| Source name |
Escherichia coli O157 TUV93-0 MEM-HEPES + DMSO
|
| Organism |
Escherichia coli |
| Characteristics |
strain: O157:H7 EHEC TUV93-0
od: 0.7
media: MEM-HEPES supplemented with 0.1% Glucose and 250nM Fe(NO3)2
no inhibitor: equivalent volume of DMSO added
|
| Treatment protocol |
Escherichia coli O157 TUV93-0 was grown in the presence of DMSO
|
| Growth protocol |
Escherichia coli O157 TUV93-0 was grown at 37 degrees C to OD 0.7 in MEM-HEPES supplemented with 0.1% Glucose and 250nM Fe(NO3)2 and and equivalent volume of DMSO.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
An RNeasy minikit was used to prepare total RNA according to the manufacturer’s instructions (QIAGEN Ltd.). Any contaminating DNA was removed using a DNAase column kit (QIAGEN Ltd).
|
| Label |
Cy3
|
| Label protocol |
using the CyScribe Post-Labelling Kit (Amersham Biosciences/GE
Healthcare) according to manufacturer’s instructions. Amino-allyllabelled
nucleotides were incorporated into the cDNA by reverse
transcription of the total RNA, followed by a direct chemical coupling
of Cy3 NHS-dye esters to the amino-allyl-labelled
cDNAs.
|
| |
|
| |
| Hybridization protocol |
Prior to hybridization, the slides were treated with the
background-reducing Pronto! Pre-Soak System (Corning Life Sciences).
The slides were incubated for 20 min in prewarmed Universal Pre-Soak
solution at 42 °C and washed twice in 0.1_SSC, 0.1% SDS for 30 s at room
temperature. Slides were immediately transferred into prewarmed prehybridization
solution (5_ SSC, 0.1% SDS, and 0.1% bovine serum albumin)
and incubated for 2–4 h at 42 °C. The microarray slides were finally washed
at room temperature once in 0.1_ SSC, 0.1% SDS for 1 min and twice in
0.1_ SSC for 30 s, briefly dipped in water and then ethanol, and dried by
centrifugation for 5 min at 800_g.
For each experiment, equal quantities (80 pmol) of each Cy5- and
Cy3-labeled cDNA were added to a final volume of 80 _l of hybridization
solution containing 25% formamide, 10 mg of bovine serum albumin
(fraction V) per ml, 5_ SSC (1_ SSC is 0.15 M NaCl, 0.015 M
sodium citrate), 0.1% SDS, 8 _g of poly(A), and 1_Denhardt’s solution.
The cDNA probes were denatured at 95 °C for 3 min and hybridized for
16 h at 42 °C. After hybridization was complete, the slides were washed
in 2_SSC, 0.1% SDS at 42 °C for 2 min in 0.1_SSC, 0.1% SDS for 2 min
at room temperature, and finally twice in 0.1_ SSC for 2 min at room
temperature. The microarray slides were dried by centrifugation for 5
min at 800g.
|
| Scan protocol |
Slide was scanned at 532 and 630 nm by using a
Genepix 4000A scanner (Axon Instruments, Union City, CA).
|
| Description |
Small molecule inhibitors have recently been shown to down regulate trascription of TTS in enterohaemorrhagic E. coli (EHEC). E. coli O157 strain TUV93-0 has been shown to induce Type III secretion (TTS) in MEM-HEPES media. Transcriptional profiling of E.coli TUV93-0 was carried out in MEM-HEPES in the prescence and absence of small molecule inhibitors to establish the global effects of these inhibitors in EHEC.
|
| Data processing |
LOWESS normalised data that represents the average of the three replicate experiments
|
| |
|
| Submission date |
Jan 30, 2008 |
| Last update date |
Aug 10, 2009 |
| Contact name |
Jai Justin Tree |
| E-mail(s) |
j.tree@unsw.edu.au
|
| Phone |
+61 2 938 59142
|
| Organization name |
University of New South Wales
|
| Department |
School of Biotechnology and Biomolecular Sciences
|
| Lab |
Tree lab
|
| Street address |
Rm s110 Bldg F25, UNSW, Gate 11 Botany St
|
| City |
Sydney |
| State/province |
NSW |
| ZIP/Postal code |
2033 |
| Country |
Australia |
| |
|
| Platform ID |
GPL3051 |
| Series (1) |
| GSE10319 |
Transcriptional response of Escherichia coli O157 TUV93-0 to small molecule inhibitors of TTS |
|
