Journal
CLINICA CHIMICA ACTA
Volume 322, Issue 1-2, Pages 113-116Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/S0009-8981(02)00161-4
Keywords
alpha-amylase; RT-PCR; isozyme expression; restriction fragment; mRNA discrimination
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Background: The alpha-amylase isozymes can be detected separately by electrophoresis; however, sometimes the identification is difficult because of their microheterogeneiety. In the present study, we tried to establish a convenient method for the detection of alpha-amylase isozyme expression. Methods: The procedure is based on three different restriction sites presented in those genes; a PstI site in both AMY 2A and 2B genes, a HaeII site in both AMY 1 and 2A genes, and a BamHI site in AMY 2B gene. After amplification from total tissue RNAs by RT-PCR with primers that were able to cover each exon, the products were cleaved with corresponding restriction endonucleases. Results: This method was applied to human samples from the parotid gland, liver (non-hepatoma), hepatoma and white blood cells (WBCs). The results indicated that the parotid gland and hepatoma (also liver) clearly expressed AMY 1 and AMY 2B genes, respectively. However, AMY 2B gene was also expressed apparently in WBCs, which produced salivary-type isozyme of the alpha-amylase, although the amylase protein was not able to identify for the hepatic isozyme. Conclusions: The method presented here might be convenient and useful for the determination of alpha-amylase isozyme expression in humans. (C) 2002 Elsevier Science B.V All rights reserved.
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