Type-2 somatostatin receptor mRNA levels in breast and colon cancer determined by a quantitative RT-PCR assay based on dual label fluorogenic probe and the TaqMan technology.
We reported previously that the expression of type 2 somatostatin receptor (sst2) was positively related to patient outcome in the childhood tumor neuroblastoma. To quantitate the expression of mRNA sst2 expression, we used a competitive RT-PCR assay. To improve the practicability of this measurement and its applicability to large groups of patients, we present here an original 'real-time' quantitative RT-PCR method, based on a dual-labeled fluorogenic probe and the TaqMan technology. By this method, we have measured sst2 mRNA expression in 24 breast cancer samples and 26 colon carcinomas as well as on the corresponding non-adjacent non-neoplastic tissue of the same patients. The proposed method has a dynamic range of 4 x 10(4) to 4 x 10(8) molecules of sst2 mRNA. The intra-assay precision of the test, evaluated as signal detection variability, was 2.4%. Accuracy, evaluated by the addition of standard RNA to unknown samples, provided a mean recovery of 98+/-2%. A significant correlation has been observed in a study performed in 24 neuroblastoma samples measured both with the proposed method and with a competitive RT-PCR assay (r=0.913, p<0.001). In our preliminary clinical study, no significant differences were observed in sst2 mRNA levels between normal and tumor specimens in both colorectal (normal tissue 5.1 x 10(7)+/-2.0 x 10(7) molecules/microg total RNA, cancer tissue 9.7 x 10(7)+/-4.2 x 10(7)) and breast tumors (normal tissue 5.5 x 10(8)+/-2.0 x 10(8), cancer tissue 4.4 x 10(8)+/-3,7 x 10(8)).However, in colorectal cancer, sst2 mRNA values of subjects with high circulating carcinoembryonic antigen (CEA) levels (>5 ng/ml) were statistically lower (2.3 x 10(7)+/-6.2 x 10(6) molecules/, microg total RNA; p<0.05) than those of subjects with low CEA concentration (1.4 x 10(8)+/-6.7 x 10(7)). Also, the sst2 mRNA ratio between normal and tumor tissue (N/T ratio) resulted significantly inversely related to CEA levels. In breast cancer, a significant difference was found between the mean N/T ratio of negative (below 10 fmol/mg protein) and positive estrogen receptor tumors (p<0.05). Analogous results were found selecting breast tumors on the basis of the progesterone receptor status (p<0.05). The proposed method is accurate, precise, sensitive and less labor-intensive than the competitive RT-PCR assay. For a correct evaluation of sst2 mRNA expression, it seems very important to measure the sst2 expression both in tumor and in the non-tumoral non-adjacent tumor specimens.
Pinzani
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Clinical Biochemistry, Department of Clinical Physiopathology, University of Florence, viale Pieraccini 6, 50139, Florence, Italy.
Orlando
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Raggi
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Distante
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Valanzano
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Tricarico
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Maggi
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Serio
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Pazzagli
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D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001483:Base Sequence; D001943:Breast Neoplasms / Q000737:chemistry / Q000235:genetics*; D002272:Carcinoembryonic Antigen / Q000378:metabolism; D015179:Colorectal Neoplasms / Q000737:chemistry / Q000235:genetics*; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D008875:Middle Aged; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000235:genetics / Q000473:pathology; D012333:RNA, Messenger / Q000378:metabolism*; D017481:Receptors, Somatostatin / Q000235:genetics* / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013194:Staining and Labeling; D019914:Taq Polymerase / Q000378:metabolism; D014407:Tumor Cells, Cultured
D002272:Carcinoembryonic Antigen; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D017481:Receptors, Somatostatin; C093733:somatostatin receptor 2; D019914:Taq Polymerase