H. pylori strain 26695 (SC#7) was grown for 15 h in BB-FBS medium containing 0.5% sodium chloride (i.e. BB-FBS-0.25%) The bacterial cells (OD600 = 0.4 to 0.5) were then centrifuged and resuspended in fresh BB-FBS-0.25% media (OD600=0.1) The bacteria were grown for 10 h, harvested by centrifugation, and frozen at -80oC
Extracted molecule
total RNA
Extraction protocol
RNA isolation was performed using the acid guanidinium-phenol-chloroform method (Trizol; Gibco BRL). Contaminating DNA was removed from the samples by treatment with RQ1 (RNA qualified) RNAse-free DNAse (Promega), and RNA was purified using the RNA Easy kit (Qiagen).
Label
33P
Label protocol
Total RNA (1 μg) was used to generate 33P-labeled cDNA using H. pylori-specific cDNA labeling primers (Sigma-Genosys). The reverse transcriptase reaction was performed for 2 h at 42o C, in the presence of 10 mM DTT, 0.33 mM dGTP, dCPT and dTTP, 5 μM dATP, 20 μCi [33P-dATP] (Perkin Elmer), 1 U RNAsin ribonuclease inhibitor (Promega) and 50 U of reverse transcriptase (Superscript II; Gibco BRL). Unincorporated 33P-dATP was removed by passage of the sample through NucAway Spin Columns (Ambion).
Hybridization protocol
The labeled cDNA was then hybridized to nylon Panorama H. pylori DNA arrays (Sigma-Genosys) using the manufacturer’s protocols. Briefly, gene arrays were prehybridized in hybridization (5 X SSPE, 2% SDS, 5X denhardts, 100 ug/ml denatured salmon sperm DNA) solution for 1 h, 65oC prior to the addition of labeled cDNA. Following hybridization (overnight) at 65oC, the membranes were washed with wash solution (0.5 X SSPE, 0.2% SDS) 3x for 20 min at 65oC.
Scan protocol
Scanning of the hybridized arrays was performed with a phosphoimager (Fuji Inc), and the hybridization signals were quantified with ImageQuant (Fuji Inc.) and Array Vision (Imaging Research Inc.) software. Array vision software is designed by Imaging Research to analyze all the spots on the H. pylori array.
Description
None
Data processing
For each array feature, a normalized signal intensity was calculated as a ratio of the signal intensity for the individual array feature divided by the total signal intensity of the entire array.
