|
| Status |
Public on Apr 03, 2011 |
| Title |
PhrB.2 transcriptional profile |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
TUV93-0 pGEM
|
| Organism |
Escherichia coli O157:H7 |
| Characteristics |
strain: O157:H7 EHEC TUV93-0 pGEM
od: 0.8
media: MEM-HEPES
|
| Treatment protocol |
10 ml of culture was mixed with 10 ml of RNAprotect bacterial reagent (QIAGEN Ltd).
|
| Growth protocol |
Sample was grown shaking at 37oC in MEM-HEPES to OD600 = 0.8.
|
| 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 pGEM::PhrB
|
| Organism |
Escherichia coli O157:H7 |
| Characteristics |
strain: O157:H7 EHEC TUV93-0 pGEM::PhrB
od: 0.8
media: MEM-HEPES
|
| Treatment protocol |
10 ml of culture was mixed with 10 ml of RNAprotect bacterial reagent (QIAGEN Ltd).
|
| Growth protocol |
Sample was grown shaking at 37oC in MEM-HEPES to OD600 = 0.8.
|
| 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 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.
|
| |
|
| |
| 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 |
The transcriptional profile of PhrB was assessed by providing EHEC O157:H7 TUV93-0 with multicopy PhrB on pGEM. RNA was extracted from late-exponential (OD 0.8) cultures grown in T3S permissive conditions (MEM-HEPES) and hybridised to UBEC 3 E. coli arrays.
|
| Data processing |
LOWESS was used to normalise data generated by GenePix using Genespring GX 7.3.1.
|
| |
|
| Submission date |
Aug 25, 2009 |
| Last update date |
Apr 03, 2011 |
| 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) |
| GSE17798 |
Transcriptional profile of EHEC O157 TUV93-0 overexpressing PsrA or PsrB |
|
