Biomedical subjects

Claudio Orlando

Publications and source records attributed to Claudio Orlando.

21 records · Page 2Linked to original sources

PubMed2003

Real-time RT-PCR for the measurement of prostate-specific antigen mRNA expression in benign hyperplasia and adenocarcinoma of prostate.

Since PSA is supposed to play an active role in the progression of prostate cancer, we applied a quantitative RT-PCR to measure the absolute levels of prostate-specific antigen (PSA) mRNA expression in benign and malignant prostatic tissue. Consecutive fine needle prostate biopsy material from 59 patients (43 with prostate adenocarcinoma and 16 with benign prostatic hyperptrophy; BPH) was used for the measurement of PSA mRNA expression. In addition, we evaluated the correlation between PSA synthesis and PSA circulating levels in the same patients. The relationship between PSA mRNA expression and histological grade was also evaluated. PSA mRNA was measured with a quantitative RT-PCR, based on the use of fluorogenic probes, according to the TaqMan reaction system. The mRNA expression for PSA in prostate adenocarcinoma biopsies was highly variable, ranging from 2 x 10(4) to 2.1 x 10(8) molecules/microg total RNA with a mean value of 2.5 x 10(7) and significantly higher (p = 0.006) than that found in BPH patients (mean: 1.3 x 10(6) and range: 6.9 x 10(2) to 8 x 10(6)). The mRNA PSA expression in needle biopsy material did not seem to be related to PSA circulating levels in prostate cancer patients (r = 0.281), whereas in BPH patients the two parameters correlated significantly (r = 0.667, p < 0.01). A reduction of PSA mRNA expression in samples with a lower grade of differentiation (Gleason score 9-10) was also observed. Even though a mean increase of PSA expression was demonstrated in cancer samples, this small difference does not confirm a significant role of PSA proteolytic activity in prostate cancer progression. In conclusion, the assay procedure we proposed represents a reliable basis for more extensive study of PSA physiopathology in prostate cancer.

Adenocarcinoma↗

PubMed2002

Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies.

Careful normalization is essential when using quantitative reverse transcription polymerase chain reaction assays to compare mRNA levels between biopsies from different individuals or cells undergoing different treatment. Generally this involves the use of internal controls, such as mRNA specified by a housekeeping gene, ribosomal RNA (rRNA), or accurately quantitated total RNA. The aim of this study was to compare these methods and determine which one can provide the most accurate and biologically relevant quantitative results. Our results show significant variation in the expression levels of 10 commonly used housekeeping genes and 18S rRNA, both between individuals and between biopsies taken from the same patient. Furthermore, in 23 breast cancers samples mRNA and protein levels of a regulated gene, vascular endothelial growth factor (VEGF), correlated only when normalized to total RNA, as did microvessel density. Finally, mRNA levels of VEGF and the most popular housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were significantly correlated in the colon. Our results suggest that the use of internal standards comprising single housekeeping genes or rRNA is inappropriate for studies involving tissue biopsies.

Biopsy↗

PubMed2002

Quantitative evaluation of somatostatin receptor subtype 2 expression in sporadic colorectal tumor and in the corresponding normal mucosa.

PURPOSE: The somatostatin (SS) receptor subtype 2 (sst2) is the principal mediator of the antiproliferative effects of SS and has the highest affinity for the commercially available SS analogues. The purpose of this study was to evaluate sst2 mRNA expression by quantitative reverse transcription-PCR (RT-PCR) in colon cancers and in corresponding normal tissues. EXPERIMENTAL DESIGN: The expression of sst2 mRNA was measured with a quantitative method based on real time RT-PCR with TaqMan assay in 100 colon cancers and in the corresponding normal tissues. In a limited number of patients, these results were compared with those obtained by in situ hybridization (n = 26) and by in vivo imaging with (111)In-pentetreotide (n = 17). RESULTS: Results obtained by quantitative RT-PCR on sst2 expression in colorectal cancer were significantly related to those obtained by in situ hybridization and (111)In-pentetreotide scintigraphy. Sst2 was expressed in all of the tumors investigated without any relationship with localization, grading, and stage of disease. Although the paired, unaffected mucosa tends to express a higher abundance of sst2 than the corresponding cancer samples, this difference did not reach a statistical significance. However, in patients with elevated carcinoembryonic antigen levels (>5 ng/ml) there was a significant loss of sst2 mRNA in the tumor when compared with its paired normal tissue. CONCLUSIONS: In this study we confirmed, by a quantitative method, that colorectal cancer does not express higher concentrations of sst2 mRNA than the corresponding unaffected tissue. Conversely, a loss of sst2 was found in patients with elevated preoperative concentrations of carcinoembryonic antigen, an unfavorable prognostic marker for colorectal cancer.

Aged↗