Quantitative Analysis of Ornithine Decarboxylase mRNA by Reverse Transcription-PCR with the LightCycler System

Abstract

Polyamines originate from the decarboxylation of ornithine, which gives rise to putrescine, the first polyamine in this metabolic pathway. The reaction is catalyzed by ornithine decarboxylase (ODC; EC 4.1.1.17), a rate-limiting enzyme in polyamine synthesis. The importance of the regulation of this pathway in colon neoplasm has been elucidated, but the application of ODC expression to clinical diagnosis as a prognostic marker has not been completely defined (1)(2)(3). To be used for this purpose, the method for measuring ODC mRNA should be sensitive, accurate, and require only small samples of colon or tumor tissue. With recent innovations in PCR technology, these analytical requirements can now be achieved (4). A new thermal cycler (LightCycler; Roche) that combines continuous fluorescence monitoring with a rapid-cycle PCR within glass capillaries is now available (5), but has not yet been used in this context. This study describes a real-time reverse transcription (RT)-PCR assay based on LightCycler technology to quantify ODC mRNA. This method was successfully applied to the measurement of ODC mRNA in tumor and nontumor tissue samples from colon carcinoma patients. We studied 18 tissue samples from nine patients with colon carcinoma (seven men and two women; mean ± SD age, 65.1 ± 7.8 years) who underwent surgical treatment. Samples of tumor tissue (T) and unaffected colon (nontumor; NT) weighing ∼30 mg were obtained from the resected surgical specimen of each patient in the operating room and were immediately immersed in liquid nitrogen and stored at −80 °C until analysis. The study protocol was approved by the Vall d′Hebron Hospital Ethics Committee. The characteristics of the patients and histologic analyses of the tumor specimens are shown in Table 1⇓ . RNA isolation was performed with the Roche isolation reagent set (cat. no. 2033674) according to the manufacturer’s instructions. Samples …