Molecular screening using non radioactive differential display technique in Malay Kelantanese patients with peptic diseases
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Date
2005
Authors
Wan Taib, Wan Rohan!
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Abstract
Peptic diseases are the most common chronic diseases of adulthood and proven to have
a substantial multifactorial inherited components. Genetic influences play some role in
the predisposition to both forms of ulcers (gastric and duodenal ulcer). A small
proportion of chronic gas~c ulcers are susceptible to be transformed into malignancy.
The possible somatic mutatio~s that ~e place have not been extensively studied. The
discovery of some genetic changes at the vicinity of the chronic benign inflammatory
lesions is important in relation to the elucidation of the carcinogenesis of gastric
cancers. The general aims of this study were to screen for differentially expressed genes
in peptic diathetic patients and to apply a technique of non radioactive differential
display analysis (DDRT-PCR). DDRT-PCR has been shown to be highly effective in
identifying sequences that are differentially expressed in various cell types and this
technique makes it possible to obtain reproducible result and efficiently identify specific
mRNAs. Twenty tissue sample biopsies of gastric mucosa of the antrum were collected
from peptic diathetic patients at Endoscopy unit. Total RNAs were extracted by using
RNA extraction kit (RNeasy Mini Kit, Qiagen). The DDRT-PCR analysis was
performed by a 2- step method which were reverse transcription and polymerase chain
reactions (RNAimage Kit 1, GenHunter). Six percent denaturing Polyacrylamide Gel
Electrophoresis (PAGE) was carried out in order to obtain the size of separation of
eDNA fragments and visualized by silver staining. Once differentially expressed
mRNAs were identified, the corresponding cDNAs were eluted from the band of the gel
and reamplified. The sequence of cDNAs were determined using an ABI Prism DNA
XVI
Sequencer. The sequences were searched for its homology using GenBank. databases
provided by National Institutes of Health (NIH, USA). Two differentially expressed
genes were identified, namely, ubiquinol-cytochrome c reductase complex (Complex
III) gene and ribosomal protein L27a gene in gastritis tissue compared to normal gastric
tissue. The expressed genes can be analyzed to determine their involvement in the
pathogenesis of peptic diathesis. The determination of these genes will be used to study
whether similar genetic derangement occur in gastric cancers in the future. This
knowledge will enhance the understanding of carcinogenesis of chronic inflammatory
lesions.
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Keywords
Malay Kelantanese , Peptic diseases