total RNA from human gastric mucos, antrum, Helicobacter pylori infected, 32years old , female
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from 20~30 mg frozen gastric biopsy samples (2 biopsies) using the RNeasy mini kit (Qiagen, Hilden, Germany). RNA samples from antrum and corpus biopsies were prepared separately. In addition to the standard procedure, Dnase I (Qiagen) was used to remove trace amount of genomic DNA
Label
33p
Label protocol
cDNA probe preparation and membrane hybridization were performed according to the respective kit manuals (R&D Systems). Briefly, 4µg of total RNA was reverse transcribed into cDNA by AMV reverse transcriptase using a human cytokine-specific primer mix in the presence of [a33P]-dCTP (2000 Ci/mmol; Amersham Pharmacia, Uppsala, Sweden) and 0.3 mM of a dATP/dGTP/dTTP mixture at 42°C for 2 hours. Labeled cDNA was subsequently separated from unincorporated nucleotides using Sephadex G-25 spin columns (R&D Systems).
Hybridization protocol
The array membranes were prehybridized at 65°C for 2 hours in hybridization solution (5X SSPE, 2% (w/v) SDS, 5X Denhardt’s reagent and 100 µg/ml sonicated, salmon DNA). Labeled probes were added to 3 ml fresh hybridization solution and allowed to hybridize to the array membranes at 65°C overnight. After hybridization, membranes were washed two times with solution 1 (0.5 x SSC and 1% SDS), twice with solution 2 (0.1 x SSC and 1% SDS) for 20 minutes at 65°C and finally exposed to a phosphorus screen for 7 days
Scan protocol
The cDNA microarrays were scanned and exported as img. and inf. files using a Fuji Bio-image Analyzer BAS2000 (Fuji Photo Film Co Ltd., Japan). The images and quantitative data of gene expression levels were analyzed using the Fuji Image Reader FLA-3000/3000G software
Description
three standards were applied. First, if the intensity of the gene was at least 2 SD above the background value, it was considered to be a genuine signal. Second, only relative changes equal to, or greater than, two-fold levels of gene expression were considered to represent up or down-regulation. In addition, if the signal intensity of a given gene was changed from negative (below the signal threshold) to positive or from positive to negative, the gene was considered being up or down regulated respectively. Finally the absolute difference in signal intensity between two samples was at least more than 2 SD of the background value
Data processing
Raw Data: raw signal output from the image analysis algorithm;Raw - Background: raw data mius the average backgroud value;VALUE: raw-backgroud, normalized by 9 housekeeping genes
