|
| Status |
Public on Sep 30, 2012 |
| Title |
H. pylori pH 7.4 rep1 |
| Sample type |
RNA |
| |
|
| Source name |
H. pylori ATCC 53504, pH 7.4, 4 hours
|
| Organism |
Helicobacter pylori NCTC 11637 = CCUG 17874 = ATCC 43504 = JCM 12093 |
| Characteristics |
ph: 7.4
|
| Treatment protocol |
Bacteria harvested from TSA plates (1 x 108) were suspended in 3mL of BHI and injected into a sterile Slide-A-Lyzer® Dialysis Chamber (Pierce), then placed in 1,500mL of BHI containing 7% horse serum, 0.25% yeast extract and 5mM urea with pH adjusted to 3.0 or 7.4 and incubated for 4 hours.
|
| Growth protocol |
H. pylori strain ATCC 43504 was used. Bacteria were grown under microaerobic conditions (5% O2, 10% CO2, 85% N2) either on Trypticase Soy Agar (TSA) plates supplemented with 5% sheep blood (Gibco) or in brain heart infusion (BHI) medium (Difco) supplemented with 7% horse serum (Gibco), 0.25% yeast extract (Difco) and Dent selective supplement (Oxoid).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, California) combined with RNeasy columns (Qiagen, Valencia, California) .
|
| Label |
Cy3
|
| Label protocol |
All total RNA samples were serially diluted to a concentration of 1000ng in 5.3uL for use with the Quick Amp Labeling kit (Agilent, Santa Clara). In preparation for the microarray, the Agilent One-Color Spike-In Kit was used as a positive control. As per the manufacturer's protocol, known synthetic RNAs were added to the sample RNA isolated from cells exposed to pH 3.0 or 7.4 and amplified and labeled using the Quick Amp Labeling kit protocol.
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| |
|
| Hybridization protocol |
To prepare the cRNA for hybridization the Agilent Fragmentation mix was used according to the Quick Amp Protocol. After a 30 minute incubation period at 60º C, 55uL of 2x GE Hybridization Buffer HI-RPM was added to each reaction tube. Samples were then vortexed and briefly spun for 1 minute at 13,000 RPM to collect the sample at the bottom of the tube. Samples were then loaded onto the microarray slides and incubated in a hybridization chamber for 17 hours at 65 º C. The preparation was then washed with Agilent’s Gene Expression Wash Buffer 1 and 2 (with Triton X) according to the protocol and scanned using the DNA Microarray Scanner11.0.2.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression after 4 hrs incubation in pH 7.4 media
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 014868_D_F_20101102) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. Data were filtered by expression (20-100%) using GeneSpring GX Software 11.5.1. All data with at least a two- fold change compared to control were subjected to a T-test. Only those genes whose expression was altered by at least 2 fold with a P-value less than 0.05 were considered to represent significantly regulated genes.
Normalized signal intensity of genes filtered by expression (20%-100%) with a fold-change greater than 2.0 and were significant after subjected to a T-test.
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| |
|
| Submission date |
Jul 19, 2012 |
| Last update date |
Sep 30, 2012 |
| Contact name |
David Scott |
| E-mail(s) |
dscott@ucla.edu
|
| Organization name |
UCLA/VA GLAHS
|
| Street address |
11301 Wilshire Blvd
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
91302 |
| Country |
USA |
| |
|
| Platform ID |
GPL15831 |
| Series (1) |
| GSE39512 |
The relationship between inhibition of gastric acid secretion and eradication of Helicobacter pylori |
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