|
| Status |
Public on Jan 30, 2007 |
| Title |
TUV93-0 B |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
TUV93-0 DMEM
|
| Organism |
Escherichia coli |
| Characteristics |
Strain: O157:H7 EHEC TUV93-0
OD: 0.6
Media: DMEM
|
| Treatment protocol |
15 ml of culture was mixed with 30 ml of RNAprotect bacterial reagent (QIAGEN Ltd).
|
| Growth protocol |
Sample was grown shaking at 37oC in DMEM to OD600 = 0.6.
|
| 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 Cy5-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 Cy5 NHS-dye esters to the amino-allyl-labelled
cDNAs.
|
| |
|
| Channel 2 |
| Source name |
TUV93-0 MEM-HEPES
|
| Organism |
Escherichia coli |
| Characteristics |
Strain: O157:H7 EHEC TUV93-0
OD: 0.6
Media: MEM, 25mM HEPES
|
| Treatment protocol |
15 ml of culture was mixed with 30 ml of RNAprotect bacterial reagent (QIAGEN Ltd).
|
| Growth protocol |
Sample was grown shaking at 37oC in DMEM to OD600 = 0.6.
|
| 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 |
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.
|
| |
|
| |
| 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 |
E. coli O157 strain TUV93-0 has been shown to secrete high levels of EspD and Tir effector proteins when cultured in MEM-HEPES, typically 5-10 fold higher than when the same strains are cultured in DMEM. Expression conditions were used that are highly selective for up-regulation of the LEE. The E. coli O157:H7 transcriptome was compared from bacteria cultured in MEM-HEPES with DMEM.
|
| Data processing |
Data was analysed using GeneSpring
|
| |
|
| Submission date |
Nov 16, 2006 |
| Last update date |
Nov 20, 2006 |
| Contact name |
Chrystala Constantinidou |
| E-mail(s) |
C.Constantinidou@bham.ac.uk
|
| Phone |
+44 (0)121 414 5564
|
| Organization name |
University of Birmingham
|
| Department |
School of Biosciences
|
| Lab |
UBEC
|
| Street address |
|
| City |
Birmingham |
| ZIP/Postal code |
B15 2TT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL3051 |
| Series (1) |
| GSE6296 |
Analysis of the exression and regulation of NleA-E in Escherichia coli O157:H7 |
|
